AMPK's role in tumor initiation and progression is controversial. Here, we provide genetic evidence that AMPK is required for metastasis in mouse models of breast cancer. In a mouse model of spontaneous breast cancer metastasis, the deletion of AMPK before and after tumor onset decreased breast cancer metastasis, and similar results were obtained after AMPK deletion in breast cancer cell lines. The deletion of AMPK induces reactive oxygen species (ROS) levels in vitro and lipid oxidation in vivo, which likely impede metastasis. Indeed, antioxidants restore the ability of AMPK-deficient tumors to metastasize. By inhibiting acetyl-coenzyme A (CoA) carboxylases 1 and 2, AMPK maintains NADPH levels by reducing NADPH consumption in fatty acid synthesis and increasing NADPH generation via fatty acid oxidation, thus increasing the dependency on auxotrophic fatty acids. Consistently, AMPK is required for the expression of the fatty acid transporter CD36 in tumors, and ectopic expression of CD36 in AMPK-deficient cells restored their ability to metastasize.

PDF file, 2386K, The effect of BIIB021 on the mRNA transcript of various KSHV genes were given in this file.

A high-fat diet (HFD) promotes metastasis through increased uptake of saturated fatty acids (SFAs). The fatty acid transporter CD36 has been implicated in this process, but a detailed understanding of CD36 function is lacking. During matrix detachment, endoplasmic reticulum (ER) stress reduces SCD1 protein, resulting in increased lipid saturation. Subsequently, CD36 is induced in a p38- and AMPK-dependent manner to promote preferential uptake of monounsaturated fatty acids (MUFAs), thereby maintaining a balance between SFAs and MUFAs. In attached cells, CD36 palmitoylation is required for MUFA uptake and protection from palmitate-induced lipotoxicity. In breast cancer mouse models, CD36-deficiency induced ER stress while diminishing the pro-metastatic effect of HFD, and only a palmitoylation-proficient CD36 rescued this effect. Finally, AMPK-deficient tumors have reduced CD36 expression and are metastatically impaired, but ectopic CD36 expression restores their metastatic potential. Our results suggest that, rather than facilitating HFD-driven tumorigenesis, CD36 plays a supportive role by preventing SFA-induced lipotoxicity.

PDF file, 985K, Effect of BIIB021 on the effect of lytic proteins (RTA and vIL-6)induced by TPA in BC-1 and BC-3 cells were given in this file.

PDF file, 3187K, The densitometric analysis of blots from figure 4B is given in this file.

Hexokinase 2 (HK2), which catalyzes the first committed step in glucose metabolism, is induced in cancer cells. HK2’s role in tumorigenesis has been attributed to its glucose kinase activity. Here, we describe a kinase independent HK2 activity, which contributes to metastasis. HK2 binds and sequesters glycogen synthase kinase 3 (GSK3) and acts as a scaffold forming a ternary complex with the regulatory subunit of protein kinase A (PRKAR1a) and GSK3β to facilitate GSK3β phosphorylation and inhibition by PKA. Thus, HK2 functions as an A-kinase anchoring protein (AKAP). Phosphorylation by GSK3β targets proteins for degradation. Consistently, HK2 increases the level and stability of GSK3 targets, MCL1, NRF2, and particularly SNAIL. In addition to GSK3 inhibition, HK2 kinase activity mediates SNAIL glycosylation, which prohibits its phosphorylation by GSK3. Finally, in mouse models of breast cancer metastasis, HK2 deficiency decreases SNAIL protein levels and inhibits SNAIL-mediated epithelial mesenchymal transition and metastasis.

Cytotoxicity assays are essential for the testing and development of novel immunotherapies for the treatment of cancer. We recently described a novel cytotoxicity assay, termed the Matador assay, which was based on marine luciferases and their engineered derivatives. In this study, we describe the development of a new cytotoxicity assay termed ‘Matador-Glo assay’ which takes advantage of a thermostable variant of Click Beetle Luciferase (Luc146-1H2). Matador-Glo assay utilizes Luc146-1H2 and D-luciferin as the luciferase-substrate pair for luminescence detection. The assay involves ectopic over-expression of Luc146-1H2 in the cytosol of target cells of interest. Upon damage to the membrane integrity, the Luc146-1H2 is either released from the dead and dying cells or its activity is preferentially measured in dead and dying cells. We demonstrate that this assay is simple, fast, specific, sensitive, cost-efficient, and not labor-intensive. We further demonstrate that the Matador-Glo assay can be combined with the marine luciferase-based Matador assay to develop a dual luciferase assay for cell death detection. Finally, we demonstrate that the Luc146-1H2 expressing target cells can also be used for in vivo bioluminescence imaging applications.

Studies in three mouse models of breast cancer identified profound discrepancies between cell-autonomous and systemic Akt1- or Akt2-inducible deletion on breast cancer tumorigenesis and metastasis. Although systemic Akt1 deletion inhibits metastasis, cell-autonomous Akt1 deletion does not. Single-cell mRNA sequencing revealed that systemic Akt1 deletion maintains the pro-metastatic cluster within primary tumors but ablates pro-metastatic neutrophils. Systemic Akt1 deletion inhibits metastasis by impairing survival and mobilization of tumor-associated neutrophils. Importantly, either systemic or neutrophil-specific Akt1 deletion is sufficient to inhibit metastasis of Akt-proficient tumors. Thus, Akt1-specific inhibition could be therapeutic for breast cancer metastasis regardless of primary tumor origin. Systemic Akt2 deletion does not inhibit and exacerbates mammary tumorigenesis and metastasis, but cell-autonomous Akt2 deletion prevents breast cancer tumorigenesis by ErbB2. Elevated circulating insulin level induced by Akt2 systemic deletion hyperactivates tumor Akt, exacerbating ErbB2-mediated tumorigenesis, curbed by pharmacological reduction of the elevated insulin.

Success of immunotherapeutic approaches using genetically engineered antibodies and T cells modified with chimeric antigen receptors (CARs) depends, among other things, on the selection of antigen binding domains with desirable expression and binding characteristics. We developed a luciferase-based assay, termed Malibu-Glo Assay, which streamlines the process of optimization of an antigen binding domain with desirable properties and allows the sensitive detection of tumor antigens. The assay involves a recombinant immunoconjugate, termed Malibu-Glo reagent, comprising an immunoglobulin or a non-immunoglobulin based antigen binding domain genetically linked to a marine luciferase. Malibu-Glo reagent can be conveniently produced in mammalian cells as a secreted protein that retains the functional activity of both the antigen binding domain and the luciferase. Moreover, crude supernatant containing the secreted Malibu-Glo reagent can directly be used for detection of cell surface antigens obviating the laborious steps of protein purification and labeling. We further demonstrate the utility of Malibu-Glo assay for the selection of optimal single chain fragment variables (scFvs) with desired affinity characteristics for incorporation into CARs. In summary, Malibu-Glo assay is a fast, simple, sensitive, specific and economical assay for antigen detection with multiple applications in the fields of antibody engineering, antibody humanization and CAR-T cell therapy.

Primary effusion lymphoma (PEL) is a subtype of non-Hodgkin lymphoma associated with infection by Kaposi sarcoma-associated herpes virus (KSHV). PEL is an aggressive disease with extremely poor prognosis when treated with conventional chemotherapy. Narciclasine, a natural product present in Amaryllidaceae family of flowering plants including daffodils, belongs to a class of molecules termed 'isocarbostyril alkaloid'. We have found that narciclasine displays preferential cytotoxicity towards PEL at low nanomolar concentrations and is approximately 10 and 100-fold more potent than its structural analogs lycoricidine and lycorine, respectively. Narciclasine arrested cell-cycle progression at the G1 phase and induced apoptosis in PEL, which is accompanied by activation of caspase-3/7, cleavage of PARP and increase in the surface expression of Annexin-V. Although narciclasine treatment resulted in a marked decrease in the expression of MYC and its direct target genes,time-course experiments revealed that MYC is not a direct target of narciclasine. Narciclasine treatment neither induces the expression of KSHV-RTA/ORF50 nor the production of infectious KSHV virions in PEL. Finally, narciclasine provides dramatic survival advantages to mice in two distinct mouse xenograft models of PEL. In conclusion, our results suggest that narciclasine could be a promising agent for the treatment of PEL.

Chimeric Antigen Receptor-T (CAR-T) cell immunotherapy has produced dramatic responses in hematologic malignancies. One of the challenges in the field is the lack of a simple assay for the detection of CARs on the surface of immune effector cells. In this study, we describe a novel luciferase-based assay, termed Topanga Assay, for the detection of CAR expression. The assay utilizes a recombinant fusion protein, called Topanga reagent, generated by joining the extra-cellular domain of a CAR-target in frame with one of the marine luciferases or their engineered derivatives. The assay involves incubation of CAR expressing cells with the Topanga reagent, a few washes and measurement of luminescence. The assay can detect CARs comprising either immunoglobulin- or non-immunoglobulin-based antigen binding domains. We further demonstrate that addition of epitope tags to the Topanga reagent not only allows its convenient one step purification but also extends its use for detection of CAR cells using flow cytometry. However, crude supernatant containing the secreted Topanga reagent can be directly used in both luminescence and flow-cytometry based assays without prior protein purification. Our results demonstrate that the Topanga assay is a highly sensitive, specific, convenient, economical and versatile assay for the detection of CARs.

A simple, accurate, sensitive and robust assay that can rapidly and specifically measure the death of target cells would have applications in many areas of biomedicine and particularly for the development of novel cellular- and immune-therapeutics. In this study, we describe a novel cytotoxicity assay, termed the Matador assay, which takes advantage of the extreme brightness, stability and glow-like characteristics of recently discovered novel marine luciferases and their engineered derivatives. The assay involves expression of a luciferase of interest in target cells in a manner so that it is preferentially retained within the healthy cells but is either released from dead and dying cells or whose activity can be preferentially measured in dead and dying cells. We demonstrate that this assay is highly sensitive, specific, rapid, and can be performed in a single-step manner without the need for any expensive equipment. We further validate this assay by demonstrating its ability to detect cytotoxicity induced by several cellular and immune-therapeutic agents including antibodies, natural killer cells, chimeric antigen receptor expressing T cells and a bispecific T cell engager.

Primary effusion lymphoma (PEL) is an aggressive type of non-Hodgkin lymphoma with extremely poor prognosis when treated with conventional chemotherapy. Kaposi sarcoma-associated herpesvirus (KSHV) is the etiologic agent of PEL. KSHV-encoded viral FLICE-inhibitory protein (vFLIP)-K13 constitutively activates the NF-κB pathway, which has been shown to be essential for the survival and proliferation of PEL cells. In a K13-isogenic cell line based screening of small molecule libraries, we have identified the natural products narciclasine and its structural analog, 7-deoxynarciclasine (found in daffodils), as having preferential cytotoxicity towards K13-expressing isogenic cell line. Further, the structurally closely related lycorine (another alkaloid found in daffodils) also exhibited similar activity. All the three compounds (Narciclasine, 7-deoxynarciclasine, and lycorine) display preferential cytotoxicity against PEL in a panel of cell lines comprising 6 hematological malignancies. Narciclasine is approximately 10 and 100-fold much effective than its structural analogs, 7-deoxynarciclasine, and lycorine, respectively. Importantly, IC50 of narciclasine in PEL cell lines (⁓10 nM) are well below the physiological concentration that was achieved in murine pharmacokinetic studies (250 nM). Narciclasine arrested cell cycle progression of PEL cells at the G0/G1 phase, with a concomitant decrease in the percentage of cells in S-phase. Further, there is a significant increase in the number of cells in sub-G0 peak, representative of apoptotic cells, which was confirmed by activation of caspase-3/7, AnnexinV staining, and PARP cleavage. Narciclasine blocked the NF-κB promoter activity in PEL cells as observed by a drastic decrease in firefly luciferase activity of PEL cells stably transduced with a lentiviral NF-κB-Luc reporter. IL-6 and IL-10 are known NF-κB targets and growth factors that are constitutively secreted by PEL, striking decrease in their secretory levels were observed upon treatment with narciclasine. Narciclasine preferentially blocked K13-induced NF-κB over the constitutive NF-κB activity in cells, which were engineered to express K13 conditionally (tet-on inducible lentiviral system). IKKγ/NEMO is essential for K13-induced NF-κB. The preference of narciclasine for K13-induced NF-κB is completely abolished upon NEMO knockout by CRISPR-Cas9 system. While probing the effect of narciclasine on the levels of putative NF-κB target genes by qRT-PCR in PEL, we found that there is a significant decrease in mRNA level of MYC, with a concomitant decrease in its protein level. Subsequently, all the direct target genes of MYC (MYB, TYRO3, TERT, PMM2, SLC19A1, and HK2) were significantly down regulated upon treatment with narciclasine. Furthermore, ectopic expression of MYC from a retroviral promoter significantly diminished the anti-proliferative effect of narciclasine and its analogs in PEL. It has been shown that inhibition of MYC is toxic to PEL and compounds that target MYC display selective activity against PEL, which explains the preferential activity of narciclasine and its analogs in PEL. MYC is required for maintenance of KSHV latency, and its down regulation results in the induction of KSHV Replication and Transcription Activator (RTA/ORF50) in PEL. However, narciclasine neither induced the expression of RTA nor the production of infectious KSHV virions in PEL. Most importantly, narciclasine doubled the survival of NSG mice bearing orthotopic - Cell Line Xenograft (CLX) and Patient Derived Xenograft (PDX) models of PEL. Finally, the IC50 of narciclasine in PBMCs from normal healthy donors is approximately 4-fold higher than the PEL cells directly isolated from the mice bearing the PDX model of the disease and PEL cell lines, indicating that there is a significant therapeutic window for narciclasine, which can be exploited for the treatment of KSHV-associated malignancies.

Chimeric antigen receptor (CAR)-based cellular therapy is a revolutionary approach to treat cancer as witnessed by recent success in clinical trials for various hematological malignancies. Currently, flow cytometry based detection of fluorochrome-tagged antibodies or proteinL that binds to the Extra-Cellular Domain (ECD) of CAR molecule are the widely used methods for the detection of CARexpression. Here, we have developed a novel luciferase based assay for detecting the expression of CAR. Our assay is accurate, highly sensitive (10-5), and has a broad linearity by taking advantage of the extreme brightness of recently discovered marine luciferases (Gluc/Nluc/Tluc16/Mluc/Loluc/Paluc/Htluc). The assay is based on recombinant fusion protein technology by fusing the ECD of a CAR target in frame with one of the marine luciferases (for detection) along with several small peptide tags -Flag/ Strep -tag II/AcV5/His (for isolation). Initially, a fusion construct was made by cloning the ECD of CD19 fused in frame with Nluc. The fusion protein was produced using 293FT cells, and tested by a simple binding assay that involved 45 minutes incubation at 4oC followed by washing and detection of luminescence. More than 103 fold increases in luminescence was observed between FMC63-CARtransduced-T/NK cells and uninfected cells or a non-specific CAR transduced cells. Essentially, identical results were obtained by replacing Nluc with other marine luciferases or by using cells transduced with five distinct CARs targeting CD19. Similar strategy was successfully applied for the specific detection of CARs targeting CD20, CD30, CD33, CD123, CD138, BCMA, and SLAM7. We also show that the small peptide tags in the fusion protein can be used for the specific isolation of CAR+ve cells using anti-tag antibodies by FACS. Additionally, we purified ECD-fusion proteins for CD19 and CD33 using Strep -Tactin protein purification columns. Purified fusion proteins were fully active, as observed by successful and specific binding to respective CAR-T/NK cells. Furthermore, direct conjugation of purified ECD-fusion proteins with fluorochromes resulted in a single-step detection and/or isolation of CAR+vecells.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a disease that remains incurable due to diagnosis at advanced stages, which renders any therapeutic intervention impracticable. The vast majority of PDAC patients develop new-onset diabetes, which often culminates in dysfunction of vital organs, leading to severe morbidity and high mortality. How PDAC development leads to diabetes is still unfolding. More than 90% of PDAC cases have an oncogenic mutation that affects the proto-oncogene Kras, mostly G12D. Using several mouse models of KrasG12D-driven PDAC, which faithfully recapitulate the human disease, we observed a massive depletion of beta cell mass, occurring even at early stages of pancreatic intraepithelial neoplasia (PanIN). This phenomenon was selective for beta cells, as PanIN or PDAC formation did not affect any other type of islet cells, including alpha and delta cells, which produce Glucagon and Somatostatin, respectively. Intriguingly, both alpha and delta cells retained their localization at the periphery of islets that are devoid of beta cells, giving rise to PanIN-like structures. Such pattern was observed when KrasG12D expression was combined with genetic inactivation of other tumor suppressor genes frequently inactivated in human PDAC, including TP53 and p16Ink4a. Lastly, we also detected depletion of beta cells and presence of remnant islets resembling PanIN in human PDAC, attesting to the clinical relevance of our findings, and further rise the attractive question of whether restoration of beta cell mass could be utilized to curb PDAC driven by oncogenic Kras. Citation Format: Thienly Nguyen, Parash Parajuli, Asrar Ahmad, Purba Singh, Sailaja Eragameddy, Gopalakrishnan Ramakrishnan, Laurence Levy, Oliver Ferrigno, Yin-Yuan Mo, Radhika Pochampally, Mohammed S Razzaque, Celine Prunier, Azeddine Atfi. Restoration of beta cell mass as a synthetic lethal strategy to overcome oncogenic Kras-driven pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr B32.

Introduction: Modificiation of T cells using CD19-specific chimeric antigen receptor (CAR) therapy has produced dramatic responses against a number of hematologic malignancies in multiple clinical trials. To date, most of the CARs studied in clinical trials are derived from mouse single chain fragment variable (scFv), which can elicit an immune response when infused into human patients and thereby can limit the persistency of CAR-T cells. Indeed, a subset of patients with limited persistency of infused CAR-Ts has been observed in clinical trials. However, this can be overcome by utilizing the humanized scFv in CAR design. Here, we constructed two new CD19-specific CARs, which are derived from the scFv of two distinct humanized CD19 antibody clones and compared them with the widely used CD19-CAR derived from a mouse scFv (FMC63).

Mutations in the tumor suppressor gene TP53 contribute to the development of approximately half of all human cancers. One mechanism by which mutant p53 (mtp53) acts is through interaction with other transcription factors, which can either enhance or repress the transcription of their target genes. Mtp53 preferentially interacts with the erythroblastosis virus E26 oncogene homologue 2 (ETS2), an ETS transcription factor, and increases its protein stability. To study the mechanism underlying ETS2 degradation, we knocked down ubiquitin ligases known to interact with ETS2. We observed that knockdown of the constitutive photomorphogenesis protein 1 (COP1) and its binding partner De-etiolated 1 (DET1) significantly increased ETS2 stability, and conversely, their ectopic expression led to increased ETS2 ubiquitination and degradation. Surprisingly, we observed that DET1 binds to ETS2 independently of COP1, and we demonstrated that mutation of multiple sites required for ETS2 degradation abrogated the interaction between DET1 and ETS2. Furthermore, we demonstrate that mtp53 prevents the COP1/DET1 complex from ubiquitinating ETS2 and thereby marking it for destruction. Mechanistically, we show that mtp53 destabilizes DET1 and also disrupts the DET1/ETS2 complex thereby preventing ETS2 degradation. Our study reveals a hitherto unknown function in which DET1 mediates the interaction with the substrates of its cognate ubiquitin ligase complex and provides an explanation for the ability of mtp53 to protect ETS2.

Mutant p53 (mtp53) is an oncogene that drives cancer cell proliferation. Here we report that mtp53 associates with the promoters of numerous nucleotide metabolism genes (NMG). Mtp53 knockdown reduces NMG expression and substantially depletes nucleotide pools, which attenuates GTP-dependent protein activity and cell invasion. Addition of exogenous guanosine or GTP restores the invasiveness of mtp53 knockdown cells, suggesting that mtp53 promotes invasion by increasing GTP. In addition, mtp53 creates a dependency on the nucleoside salvage pathway enzyme deoxycytidine kinase for the maintenance of a proper balance in dNTP pools required for proliferation. These data indicate that mtp53-harbouring cells have acquired a synthetic sick or lethal phenotype relationship with the nucleoside salvage pathway. Finally, elevated expression of NMG correlates with mutant p53 status and poor prognosis in breast cancer patients. Thus, mtp53's control of nucleotide biosynthesis has both a driving and sustaining role in cancer development.