Indranil Basu

A Transition State Analogue of 5′-Methylthioadenosine Phosphorylase Induces Apoptosis in Head and Neck Cancers

Methylthio-DADMe-immucillin-A (MT-DADMe-ImmA) is an 86-pm inhibitor of human 5′-methylthioadenosine phosphorylase (MTAP). The sole function of MTAP is to recycle 5′-methylthioadenosine (MTA) to S-adenosylmethionine. Treatment of cultured cells with MT-DADMe-ImmA and MTA inhibited MTAP, increased cellular MTA concentrations, decreased polyamines, and induced apoptosis in FaDu and Cal27, two head and neck squamous cell carcinoma cell lines. The same treatment did not induce apoptosis in normal human fibroblast cell lines (CRL2522 and GM02037) or in MCF7, a breast cancer cell line with an MTAP gene deletion. MT-DADMe-ImmA alone did not induce apoptosis in any cell line, implicating MTA as the active agent. Treatment of sensitive cells caused loss of mitochondrial inner membrane potential, G2/M arrest, activation of mitochondria-dependent caspases, and apoptosis. Changes in cellular polyamines and MTA levels occurred in both responsive and nonresponsive cells, suggesting cell-specific epigenetic effects. A survey of aberrant DNA methylation in genomic DNA using a microarray of 12,288 CpG island clones revealed decreased CpG island methylation in treated FaDu cells compared with untreated cells. FaDu tumors in a mouse xenograft model were treated with MT-DADMe-ImmA, resulting in tumor remission. The selective action of MT-DADMe-ImmA on head and neck squamous cell carcinoma cells suggests potential as an agent for treatment of cancers sensitive to reduced CpG island methylation.

Growth and Metastases of Human Lung Cancer Are Inhibited in Mouse Xenografts by a Transition State Analogue of 5′-Methylthioadenosine Phosphorylase

The S-adenosylmethionine (AdoMet) salvage enzyme 5′-methylthioadenosine phosphorylase (MTAP) has been implicated as both a cancer target and a tumor suppressor. We tested these hypotheses in mouse xenografts of human lung cancers. AdoMet recycling from 5′-methylthioadenosine (MTA) was blocked by inhibition of MTAP with methylthio-DADMe-Immucillin-A (MTDIA), an orally available, nontoxic, picomolar transition state analogue. Blood, urine, and tumor levels of MTA increased in response to MTDIA treatment. MTDIA treatment inhibited A549 (human non-small cell lung carcinoma) and H358 (human bronchioloalveolar non-small cell lung carcinoma cells) xenograft tumor growth in immunodeficient Rag2−/−γC−/− and NCr-nu mice. Systemic MTA accumulation is implicated as the tumor-suppressive metabolite because MTDIA is effective for in vivo treatment of A549 MTAP−/− and H358 MTAP+/+ tumors. Tumors from treated mice showed increased MTA and decreased polyamines but little alteration in AdoMet, methionine, or adenine levels. Gene expression profiles of A549 tumors from treated and untreated mice revealed only modest alterations with 62 up-regulated and 63 down-regulated mRNAs (≥3-fold). MTDIA antitumor activity in xenografts supports MTAP as a target for lung cancer therapy.

Induction of Androgen Receptor-Dependent Apoptosis in Prostate Cancer Cells by the Retinoblastoma Protein

Re-expression of a tumor suppressor in tumor cells that lack it is an effective way to study its functional activities. However, because tumor cells contain multiple mutations, tumor suppressor functions that are dependent on (an)other regulators are unlikely to be identified by its re-expression alone if the other regulators are also mutated. In this study, we show that re-expression of retinoblastoma (RB) together with the androgen receptor (AR) in RB- and AR-deficient prostate cancer DU-145 cells resulted in an apoptotic activity, acting through the mitochondria damage-initiated caspase activation pathway, which was not present when RB, or the AR, was re-expressed alone. The ability of RB + AR to induce mitochondria damage was dependent on the proapoptotic proteins Bax and Bak and could be blocked by the antiapoptotic protein Bcl-xL. Coexpressed AR did not detectably change RB’s regulation of E2F and cell cycle progression in culture. On the other hand, coexpressed RB could activate the transactivation activity of the AR in an androgen-depleted media. Although androgen induced greater AR transactivation activity in this condition, it did not induce apoptosis in the absence of coexpressed RB. Analysis of mutants of RB and the AR indicated that intact pocket function of RB and the transactivation activity of the AR were required for RB + AR-induced apoptosis. These results provide direct functional data for an AR-dependent apoptosis-inducing activity of RB and highlight the importance of cell type-specific regulators in obtaining a more complete understanding of RB.

Rigosertib Is a More Effective Radiosensitizer Than Cisplatin in Concurrent Chemoradiation Treatment of Cervical Carcinoma, In Vitro and In Vivo

PURPOSE To compare rigosertib versus cisplatin as an effective radiosensitizing agent for cervical malignancies. METHODS AND MATERIALS Rigosertib and cisplatin were tested in cervical cancer cell lines, HeLa and C33A. A 24-hour incubation with rigosertib and cisplatin, before irradiation (2-8 Gy), was used for clonogenic survival assays. Cell cycle analysis (propidium iodide staining) and DNA damage (γ-H2AX expression) were evaluated by fluorescence-activated cell sorter cytometry. Rigosertib was also tested in vivo in tumor growth experiments on cervical cancer xenografts. RESULTS Rigosertib was demonstrated to induce a G2/M block in cancer cells. Survival curve comparison revealed a dose modification factor, as index of radiosensitization effect, of 1.1-1.3 for cisplatin and 1.4-2.2 for rigosertib. With 6-Gy irradiation, an increase in DNA damage of 15%-25% was achieved in both HeLa and C33A cells with cisplatin pretreatment, and a 71-108% increase with rigosertib pretreatment. In vivo tumor growth studies demonstrated higher performance of rigosertib when compared with cisplatin, with 53% longer tumor growth delay. CONCLUSIONS Rigosertib was more effective than cisplatin when combined with radiation and caused minimal toxicity. These data support the need for clinical trials with rigosertib in combination therapy for patients with cervical carcinoma.

Structure-guided development of a high-affinity human Programmed Cell Death-1: Implications for tumor immunotherapy

Programmed Cell Death-1 (PD-1) is an inhibitory immune receptor, which plays critical roles in T cell co-inhibition and exhaustion upon binding to its ligands PD-L1 and PD-L2. We report the crystal structure of the human PD-1 ectodomain and the mapping of the PD-1 binding interface. Mutagenesis studies confirmed the crystallographic interface, and resulted in mutant PD-1 receptors with altered affinity and ligand-specificity. In particular, a high-affinity mutant PD-1 (HA PD-1) exhibited 45 and 30-fold increase in binding to PD-L1 and PD-L2, respectively, due to slower dissociation rates. This mutant (A132L) was used to engineer a soluble chimeric Ig fusion protein for cell-based and in vivo studies. HA PD-1 Ig showed enhanced binding to human dendritic cells, and increased T cell proliferation and cytokine production in a mixed lymphocyte reaction (MLR) assay. Moreover, in an experimental model of murine Lewis lung carcinoma, HA PD-1 Ig treatment synergized with radiation therapy to decrease local and metastatic tumor burden, as well as in the establishment of immunological memory responses. Our studies highlight the value of structural considerations in guiding the design of a high-affinity chimeric PD-1 Ig fusion protein with robust immune modulatory properties, and underscore the power of combination therapies to selectively manipulate the PD-1 pathway for tumor immunotherapy.

Low-Intensity Focused Ultrasound Induces Reversal of Tumor-Induced T Cell Tolerance and Prevents Immune Escape

Immune responses against cancer cells are often hindered by immunosuppressive mechanisms that are developed in the tumor microenvironment. Induction of a hyporesponsive state in tumor Ag-specific T cells is one of the major events responsible for the inability of the adaptive immune system to mount an efficient antitumor response and frequently contributes to lessen the efficacy of immunotherapeutic approaches. Treatment of localized tumors by focused ultrasound (FUS) is a minimally invasive therapy that uses a range of input energy for in situ tumor ablation through the generation of thermal and cavitation effect. Using a murine B16 melanoma tumor model, we show that a variant of FUS that delivers a reduced level of energy at the focal point and generates mild mechanical and thermal stress in target cells has the ability to increase immunogenic presentation of tumor Ags, which results in reversal of tumor-induced T cell tolerance. Furthermore, we show that the combination of nonablative low-energy FUS with an ablative hypofractionated radiation therapy results in synergistic control of primary tumors and leads to a dramatic reduction in spontaneous pulmonary metastases while prolonging recurrence-free survival only in immunocompetent mice.

Immunomodulation of prostate cancer cells after low energy focused ultrasound

High-Intensity Focused Ultrasound (HIFU) is a promising non-invasive treatment for localized solid tumors. While patients treated with HIFU show symptomatic improvement, the majority die from local recurrence and metastases due to incomplete tumor ablation and inability to control spread outside the primary tumor site. Therefore, improved local control and a concomitant systemic therapeutic effect would be valuable for successful tumor control by HIFU. Low intensity focused ultrasound (LOFU) induces sonic stress by raising the temperature without killing the cells. We demonstrated that tumor pre-treatment with LOFU prior to HIFU results in tumor growth retardation (Figure ​(Figure1A)1A) and induces a Th1 predominant immune response (Figure ​(Figure1B).1B). To further validate the immunological consequences of LOFU pre-treatment, we investigated the immunomodulation of prostate cancer caused by LOFU. We previously demonstrated that LOFU induced expression of genes related to the unfolded protein response (UPR) and endoplasmic reticulum (ER) stress. We hypothesize that LOFU increases immunomodulatory surface signals, such as heat shock protein 70 (HSP70) and calreticulin. HSP70 is an endogenous “danger” signal, which can activate dendritic cells. Calreticulin is an “eat me” signal, which encourages phagocytosis and thus antigen processing for presentation on cell surface MHC for T cell recognition. Thus, sequential administration of LOFU and HIFU provides a source of tumor antigens and endogenous “danger” signals for dendritic cell (DC) activation, thereby inducing a tumor-specific systemic immune response that augments the efficacy of therapeutic ultrasound to control both local and systemic disease. Figure 1 A. Tumor Growth Retardation. B. LOFU+HIFU induced tumor-specific T cell response. C. Flow cytometry of HSP70 and calreticulin of LOFU-treated prostate cancer cells. D. IF of RM-1 and PC3 cells stained for HSP70 Methods LOFU treatment was performed on the Philips Therapy and Imaging Probe System (TIPS, Philips Research Briarcliff, USA) using 3W, 100% duty cycle, 1.5 seconds, 1 mm spacing. For IF analysis, LOFU-treated cells were cytospined and fixed with 4% paraformaldehyde and stained with rabbit ant-HSP70 overnight, followed by incubation with a secondary goat anti-rabbit PE conjugated and anti-phalloidin antibodies for 1 hour at room temperature. DAPI was included in the mounting medium and slides were analyzed using an Inverted Olympus IX81. Flow cytometry staining included a live/dead cell marker to isolate only living cells expressing HSP70 on the cell surface.

In vitro comparative efficacy of ON01910.Na and cisplatin with radiation on cervical carcinoma cells

16565 Background: A synthetic benzyl styryl sulfone (ON019010.Na) has been shown to have anti-tumor activity, alone and in combination with other chemotherapeutic agents as a Chk2 inhibitor and ant...