Aadra P. Bhatt

Dual inhibition of PI3K and mTOR inhibits autocrine and paracrine proliferative loops in PI3K/Akt/mTOR-addicted lymphomas

Primary effusion lymphoma (PEL) constitutes a subset of non-Hodgkin lymphoma whose incidence is highly increased in the context of HIV infection. Kaposi sarcoma-associated herpesvirus is the causative agent of PEL. The phosphatidylinositol 3-kinase (PI3K) signaling pathway plays a critical role in cell proliferation and survival, and this pathway is dysregulated in many different cancers, including PEL, which display activated PI3K, Akt, and mammalian target of rapamycin (mTOR) kinases. PELs rely heavily on PI3K/Akt/mTOR signaling, are dependent on autocrine and paracrine growth factors, and also have a poor prognosis with reported median survival times of less than 6 months. We compared different compounds that inhibit the PI3K/Akt/mTOR pathway in PEL. Although compounds that modulated activity of only a single pathway member inhibited PEL proliferation, the use of a novel compound, NVP-BEZ235, that dually inhibits both PI3K and mTOR kinases was significantly more efficacious in culture and in a PEL xenograft tumor model. NVP-BEZ235 was effective at low nanomolar concentrations and has oral bioavailability. We also report a novel mechanism for NVP-BEZ235 involving the suppression of multiple autocrine and paracrine growth factors required for lymphoma survival. Our data have broad applicability for the treatment of cytokine-dependent tumors with PI3K/mTOR dual inhibitors.

Dysregulation of fatty acid synthesis and glycolysis in non-Hodgkin lymphoma

The metabolic differences between B-NHL and primary human B cells are poorly understood. Among human B-cell non-Hodgkin lymphomas (B-NHL), primary effusion lymphoma (PEL) is a unique subset that is linked to infection with Kaposis sarcoma-associated herpesvirus (KSHV). We report that the metabolic profiles of primary B cells are significantly different from that of PEL. Compared with primary B cells, both aerobic glycolysis and fatty acid synthesis (FAS) are up-regulated in PEL and other types of nonviral B-NHL. We found that aerobic glycolysis and FAS occur in a PI3K-dependent manner and appear to be interdependent. PEL overexpress the fatty acid synthesizing enzyme, FASN, and both PEL and other B-NHL were much more sensitive to the FAS inhibitor, C75, than primary B cells. Our findings suggest that FASN may be a unique candidate for molecular targeted therapy against PEL and other B-NHL.

AKTivation of PI3K/AKT/mTOR signaling pathway by KSHV

As an obligate intracellular parasite, Kaposi sarcoma-associated herpesvirus (KSHV) relies on the host cell machinery to meet its needs for survival, viral replication, production, and dissemination of progeny virions. KSHV is a gammaherpesvirus that is associated with three different malignancies: Kaposi sarcoma (KS), and two B cell lymphoproliferative disorders, primary effusion lymphoma (PEL) and multicentric Castleman’s disease. KSHV viral proteins modulate the cellular phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway, which is a ubiquitous pathway that also controls B lymphocyte proliferation and development. We review the mechanisms by which KSHV manipulates the PI3K/AKT/mTOR pathway, with a specific focus on B cells.

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Rapalogs in viral cancers

At present, 150 clinical trials are registered with the National Cancer Institute, which investigate the efficacy of inhibitors of the PI3K/Akt/mTOR pathway against multiple cancers. Efficacy varies not so much with drug action, but with tumor type, as different cancer types (and different pre-clinical models) exhibit widely differing susceptibilities to mTOR inhibitors, such as rapamycin. Viral cancers appear to be among the most mTOR-addicted and most rapamycin-sensitive cancers. We discuss the different mTOR inhibitors that are currently available and in clinical trials. We also speculate how the molecular makeup of viral cancers could guide the selection and use of known and novel mTOR inhibitors to treat virus-associated malignancies.

Regulation of drug metabolism and toxicity by multiple factors of genetics, epigenetics, lncRNAs, gut microbiota, and diseases: a meeting report of the 21st International Symposium on Microsomes and Drug Oxidations (MDO)

Variations in drug metabolism may alter drug efficacy and cause toxicity; better understanding of the mechanisms and risks shall help to practice precision medicine. At the 21st International Symposium on Microsomes and Drug Oxidations held in Davis, California, USA, in October 2–6, 2016, a number of speakers reported some new findings and ongoing studies on the regulation mechanisms behind variable drug metabolism and toxicity, and discussed potential implications to personalized medications. A considerably insightful overview was provided on genetic and epigenetic regulation of gene expression involved in drug absorption, distribution, metabolism, and excretion (ADME) and drug response. Altered drug metabolism and disposition as well as molecular mechanisms among diseased and special populations were presented. In addition, the roles of gut microbiota in drug metabolism and toxicology as well as long non-coding RNAs in liver functions and diseases were discussed. These findings may offer new insights into improved understanding of ADME regulatory mechanisms and advance drug metabolism research.

A viral kinase mimics S6 kinase to enhance cell proliferation

Significance Viruses usurp the host cell machinery to replicate, disseminate, and propagate themselves. Kaposi’s sarcoma-associated herpesvirus (KSHV) encodes a viral protein kinase (vPK) also known as ORF36. Using in silico modeling and biochemistry, we report that vPK/ORF36 displays limited homology to cellular S6 kinase B1 (S6KB1). Both kinases share overlapping substrates and can phosphorylate S6. However, unlike S6KB1, vPK augments S6 phosphorylation under conditions where mammalian target of rapamycin (mTOR) is inhibited. vPK modulates cellular proliferation and protein synthesis, augments anchorage independence, and enhances angiogenesis. Depletion of vPK/ORF36 during lytic replication inhibits the production of infectious virions, thereby underscoring the importance of this kinase during the KSHV life cycle. Our collective observations suggest that vPK may function as a constitutively active mimic of S6KB1. Viruses depend upon the host cell for manufacturing components of progeny virions. To mitigate the inextricable dependence on host cell protein synthesis, viruses can modulate protein synthesis through a variety of mechanisms. We report that the viral protein kinase (vPK) encoded by open reading frame 36 (ORF36) of Kaposi’s sarcoma-associated herpesvirus (KSHV) enhances protein synthesis by mimicking the function of the cellular protein S6 kinase (S6KB1). Similar to S6KB1, vPK phosphorylates the ribosomal S6 protein and up-regulates global protein synthesis. vPK also augments cellular proliferation and anchorage-independent growth. Furthermore, we report that both vPK and S6KB1 phosphorylate the enzyme 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 2 (PFKFB2) and that both kinases promote endothelial capillary tubule formation.

Human herpesvirus–encoded kinase induces B cell lymphomas in vivo

Kaposi’s sarcoma–associated herpesvirus (KSHV) is a gammaherpesvirus that is the etiological agent of the endothelial cell cancer Kaposi’s sarcoma (KS) and 2 B cell lymphoproliferative disorders, primary effusion lymphoma (PEL) and multicentric Castleman’s disease (MCD). KSHV ORF36, also known as viral protein kinase (vPK), is a viral serine/threonine kinase. We previously reported that KSHV vPK enhances cell proliferation and mimics cellular S6 kinase to phosphorylate ribosomal protein S6, a protein involved in protein synthesis. We created a mouse model to analyze the function of vPK in vivo. We believe this is the first mouse tumor model of a viral kinase encoded by a pathogenic human virus. We observed increased B cell activation in the vPK transgenic mice compared with normal mice. We also found that, over time, vPK transgenic mice developed a B cell hyperproliferative disorder and/or a high-grade B cell non-Hodgkin lymphoma at a greatly increased incidence compared with littermate controls. This mouse model shows that a viral protein kinase is capable of promoting B cell activation and proliferation as well as augmenting lymphomagenesis in vivo and may therefore contribute to the development of viral cancers.

Nonsteroidal Anti-Inflammatory Drug-Induced Leaky Gut Modeled Using Polarized Monolayers of Primary Human Intestinal Epithelial Cells

The intestinal epithelium provides a critical barrier that separates the gut microbiota from host tissues. Nonsteroidal anti-inflammatory drugs (NSAIDs) are efficacious analgesics and antipyretics and are among the most frequently used drugs worldwide. In addition to gastric damage, NSAIDs are toxic to the intestinal epithelium, causing erosions, perforations, and longitudinal ulcers in the gut. Here, we use a unique in vitro human primary small intestinal cell monolayer system to pinpoint the intestinal consequences of NSAID treatment. We found that physiologically relevant doses of the NSAID diclofenac (DCF) are cytotoxic because they uncouple mitochondrial oxidative phosphorylation and generate reactive oxygen species. We also find that DCF induces intestinal barrier permeability, facilitating the translocation of compounds from the luminal to the basolateral side of the intestinal epithelium. The results we outline here establish the utility of this novel platform, representative of the human small intestinal epithelium, to understand NSAID toxicity, which can be applied to study multiple aspects of gut barrier function including defense against infectious pathogens and host-microbiota interactions.

Abstract B39: Multitarget approach against PI3K, Aurora kinase, and BRD4 leads to improved antitumor activity in Myc-overexpressing lymphoma cells

Background: Myc has proven extremely difficult to target therapeutically. Therefore, we hypothesized that optimal inhibition of several key targetable pathways involved in Myc signaling could overcome this long-standing problem. We identified phosphoinositide 3-kinase (PI3K), aurora kinase (Aurk), and bromodomain protein (BRD)4 as the primary therapeutic targets to counteract Myc deregulation based on strong evidence that these pathways are essential for tumor maintenance in Myc-driven malignancies. Methods: Cytotoxicity assays using MTS and trypan blue were used to compare levels of drug sensitivity in lymphoma cell lines with high and low Myc mRNA expression. Apoptosis and cell cycle assays were performed using Annexin V and Propidium Iodide staining. Murine xenograft models were used to assess the efficacy and tolerability of single vs. combined inhibition. Results: Myc-overexpressing Burkitt lymphoma (Raji) cells were treated with various concentrations of small molecule inhibitors against PI3K (BEZ-235), Aurk (MLN-8237), or BRD4 (I-BET-151) for 48 to 72 hours and cell viability was evaluated. BEZ-235, MLN-8237, and I-BET-151 inhibited cell growth individually with IC-50 of 30 nM, 10 nM, and 650 nM, respectively. Dual treatment with BEZ-235/MLN-8237, BEZ-235/I-BET-151, or MLN-8237/I-BET-151 induced more significant cell growth inhibition as compared to treatment with the single agent alone. The combination index (CI) values were less than 1 at various drug concentrations, indicating that different combinations of BEZ-235, MLN-8237, and I-BET-151 were synergistic in terms of inhibitory effect on tumor cell viability. Superior activity of the dual inhibition was also noted in other Myc-overexpressing lymphoma cells, including Ramos and SUDHL4. Combination treatments also increased apoptosis and induced more pronounced cell cycle arrest compared to the single agent treatment alone. We then analyzed protein expression by Western blot in Myc-overexpressing cells treated with various combinations of BEZ-235, MLN-8237, and I-BET-151. Treatment with BEZ-235 in Myc-overexpressing lymphoma cells resulted in reduced phosphorylated levels of the downstream effector RPS6K, which promotes protein translation and proliferation. MLN-8237 reduced the expression of p-HisH3 and p-Aurk while increasing the expression of p-S6K. Treatment with I-BET-151 resulted in significant reduction of Myc expression. Combined treatments had minimal impact on protein expression patterns compared to individual treatments, and the synergistic effect was independent of depletion of cytoplasmic levels of Myc. Lastly, athymic nude mice bearing Ramos lymphoma xenografts were treated with BEZ-235, MLN-8237, I-BET-151, and various dual-combinations of each agent. The mean tumor volumes in mice treated with negative control, BEZ-235, I-BET-151, and MLN-8237 as single agents were 3480, 2364, 2320, and 671 mm3, respectively, at Day 28. Mice treated with BEZ-235/I-BET-151, MLN-8237/I-BET-151, and BEZ-235/MLN-8237 combinations had mean tumor volumes of 1709, 461, and 166 mm3, respectively, at Day 28. The survival rates for mice treated with negative control, BEZ-235, I-BET-151, and MLN-8237 as single agents were 0%, 10%, 10%, and 70%, respectively, at Day 35. The combination of BEZ-235 and MLN-8237 was associated with significant toxicity with 60% of mice dying from weight loss and failure to thrive despite tumor regression. Mice treated with the MLN-8237 and I-BET-151 combination demonstrated the best survival rate of 100% at Day 35. Conclusion: Our data demonstrated that Myc-overexpressing tumors can be successfully targeted by inhibiting kinases associated with Myc-signaling. Specifically, MLN-8237, a small molecule inhibitor against AURK, induced apoptosis of Myc-overexpressing tumor cells in vitro and showed the most promising anti-tumor activity in mice bearing Myc-overexpressing lymphoma, especially when combined with I-BET-151, a BRD4 inhibitor. Citation Format: Steven I. Park, Carolina P. Lin, Michael Foote, Trevor Parton, David B. Darr, Daniel Roth, Aadra P. Bhatt, Dirk P. Dittmer, Norman E. Sharpless, Blossom Damania. Multitarget approach against PI3K, Aurora kinase, and BRD4 leads to improved antitumor activity in Myc-overexpressing lymphoma cells. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B39.