Gain-of-function mutations in the catalytic site of EZH2 (Enhancer of Zeste Homologue 2), is observed in about 22% of diffuse large B-cell lymphoma (DLBCL) cases. Here we show that selective inhibition of histone deacetylase 1,2 (HDAC1,2) activity using a small molecule inhibitor causes cytotoxic or cytostatic effects in EZH2 gain-of-function mutant (EZH2GOF) DLBCL cells. Our results show that blocking the activity of HDAC1,2 increases global H3K27ac without causing a concomitant global decrease in H3K27me3 levels. Our data shows that inhibition of HDAC1,2 is sufficient to decrease H3K27me3 present at DSBs, decrease DSB repair and activate the DNA damage response in these cells. In addition to increased H3K27me3, we found that the EZH2GOF DLBCL cells overexpress another chemotherapy resistance factor − B-lymphoma and BAL-associated protein (BBAP). BBAP monoubiquitinates histone H4K91, a residue that is also subjected to acetylation. Our results show that selective inhibition of HDAC1,2 increases H4K91ac, decreases BBAP-mediated H4K91 monoubiquitination, impairs BBAP-dependent DSB repair and sensitizes the refractory EZH2GOF DLBCL cells to treatment with doxorubicin, a chemotherapy agent. Hence, selective HDAC1,2 inhibition provides a novel DNA repair mechanism-based therapeutic approach as it can overcome both EZH2- and BBAP-mediated DSB repair in the EZH2GOF DLBCL cells.

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Despite increasing use of "targeted therapy," interleukin-2 (IL-2) is unique, because this cytokine can induce long-term remissions in 5% to 7% of patients with metastatic melanoma and renal cancer. Clinical use of IL-2 is limited by severe toxicities, such as hypotension and vascular leak syndrome (VLS). Nitric oxide seems to be involved in the pathogenesis of these toxicities. On the basis of previous studies, we hypothesized that the endothelial nitric oxide synthase (eNOS) is the major source of nitric oxide. Mice with a knockout of the eNOS isoenzyme were treated with IL-2 (800,000 IU twice daily for 5 d). Blood pressure and vascular leak were measured. Inhibitors of superoxide, nitric oxide, and soluble guanylate cyclase were used to probe the mechanism. These experiments showed that IL-2 treatment increased eNOS messenger ribonucleic acid expression and nitric oxide metabolite excretion in eNOS knockout mice. Unlike normal and inducible nitric oxide synthase knockout mice, eNOS knockout mice proved resistant to IL-2-induced hypotension and vascular leak. Although hypotension seems to be mediated by superoxide or peroxynitrite, vascular leak seemed to be mediated by nitric oxide. Inhibition of guanylate cyclase and cyclic guanylate monophosphate formation during IL-2 treatment using methylene blue (MB)-inhibited vascular leak. MB treatment did not interfere with IL-2-induced antitumor mechanisms. Our experiments established that eNOS is a key mediator of IL-2-induced VLS and hypotension. A clinical trial of MB infusion during IL-2 therapy is currently being planned.

Circulating tumor cells (CTC) are rarely detected in the blood of cancer patients, even though they are a direct harbinger of eventual patient demise. We developed an innovative CTC culture technology to allow more sensitive isolation, expansion, and characterization of viable colonies from patient blood. In this assay, the entire leukocyte fraction from 10 ml of anticoagulated patient blood is placed into culture medium without any pre-selection. After 16 days in culture, CTC derived colonies are counted. As a proof-of-principle, blood samples from 58 Stage IIa-IV melanoma patients were tested. Ninety percent of these samples grew colonies. The colony numbers ranged from 0 -308 (mean 63 ± 9.5 SEM). Ten normal volunteers had virtually no growth (mean 0.5 ± 1.4 colonies). Colonies were harvested using a micropipette for characterization. Tumor-cell containing spheroids were embedded in paraffin, sectioned, and stained with melanoma-specific mAb for histologic characterization. MITF proved to be the most consistent immunostain that identified melanoma cells in these colonies. A host-cell component in colonies was also identified using CD68 and CD43 mAb staining. Following enzymatic dissociation of colonies, a variety of immunostains were tested. Papanicolau staining proved most useful for identifying the abnormal nuclei of tumor cells. Flow cytometry could readily distinguish host and tumor cell populations based on DNA content and forward/side scatter in dissociated colonies. The stem cell marker ALDH1A1 associated with the aneuploid population, but CD45 was expressed on both diploid and aneuploid cells. 1468The ability to repeatedly isolate CTC derived colonies from cancer patient blood samples opens the door to a novel type of long-term clinical monitoring. This novel CTC culture technology may prove useful to perform molecular characterization, assessment of treatment response, and testing of drug sensitivity and resistance in patients during treatment.