Dheeraj Pelluru

Elevated IL-17 produced by Th17 cells promotes myeloma cell growth and inhibits immune function in multiple myeloma

Elevated cytokines in bone marrow (BM) micro-environment (interleukin-6 [IL-6], transforming growth factor-beta [TGF-beta], and IL-1beta) may play an important role in observed immune dysfunction in multiple myeloma (MM). As IL-6 and TGF-beta are important for the generation of T-helper 17 (T(H)17) cells, we evaluated and observed a significantly elevated baseline and induced frequency of T(h)17 cells in peripheral blood mononuclear cells (PBMCs) and BM mononuclear cells (BMMCs) from MM patients compared with healthy donors. We observed significant increase in levels of serum IL-17, IL-21, IL-22, and IL-23 in blood and BM in MM compared with healthy donors. We also observed that myeloma PBMCs after T(H)17 polarization significantly induced IL-1alpha, IL-13, IL-17, and IL-23 production compared with healthy donor PBMCs. We next observed that IL-17 promotes myeloma cell growth and colony formation via IL-17 receptor, adhesion to bone marrow stromal cells (BMSCs) as well as increased growth in vivo in murine xenograft model of human MM. Additionally, we have observed that combination of IL-17 and IL-22 significantly inhibited the production of T(H)1-mediated cytokines, including interferon-gamma (IFN-gamma), by healthy donor PBMCs. In conclusion, IL-17-producing T(h)17 cells play an important role in MM pathobiology and may be an important therapeutic target for anti-MM activity and to improve immune function.

The sumoylation pathway is dysregulated in multiple myeloma and is associated with adverse patient outcome

Multiple myeloma (MM) is a plasma cell neoplasm that proceeds through a premalignant state of monoclonal gammopathy of unknown significance; however, the molecular events responsible for myelomagenesis remain uncharacterized. To identify cellular pathways deregulated in MM, we addressed that sumoylation is homologous to ubiquitination and results in the attachment of the ubiquitin-like protein Sumo onto target proteins. Sumoylation was markedly enhanced in MM patient lysates compared with normal plasma cells and expression profiling indicated a relative induction of sumoylation pathway genes. The Sumo-conjugating enzyme Ube2I, the Sumo-ligase PIAS1, and the Sumo-inducer ARF were elevated in MM patient samples and cell lines. Survival correlated with expression because 80% of patients with low UBE2I and PIAS1 were living 6 years after transplantation, whereas only 45% of patients with high expression survived 6 years. UBE2I encodes the sole Sumo-conjugating enzyme in mammalian cells and cells transfected with a dominant-negative sumoylation-deficient UBE2I mutant exhibited decreased survival after radiation exposure, impaired adhesion to bone marrow stroma cell and decreased bone marrow stroma cell-induced proliferation. UBE2I confers cells with multiple advantages to promote tumorigenesis and predicts decreased survival when combined with PIAS1. The sumoylation pathway is a novel therapeutic target with implications for existing proteasomal-based treatment strategies.

Targeting IL-17A in multiple myeloma: a potential novel therapeutic approach in myeloma.

We have previously demonstrated that interleukin-17A (IL-17) producing T helper 17 cells are significantly elevated in blood and bone marrow (BM) in multiple myeloma (MM) and IL-17A promotes MM cell growth via the expression of IL-17 receptor. In this study, we evaluated anti-human IL-17A human monoclonal antibody (mAb), AIN457 in MM. We observe significant inhibition of MM cell growth by AIN457 both in the presence and the absence of BM stromal cells (BMSCs). Although IL-17A induces IL-6 production, AIN457 significantly downregulated IL-6 production and MM cell adhesion in MM–BMSC co-culture. AIN457 also significantly inhibited osteoclast cell differentiation. More importantly, in the SCIDhu model of human myeloma administration of AIN457 weekly for 4 weeks after the first detection of tumor in mice led to a significant inhibition of tumor growth and reduced bone damage compared with isotype control mice. To understand the mechanism of action of anti-IL-17A mAb, we report, here, that MM cells express IL-17A. We also observed that IL-17A knockdown inhibited MM cell growth and their ability to induce IL-6 production in co-cultures with BMSC. These pre-clinical observations suggest efficacy of AIN457 in myeloma and provide the rationale for its clinical evaluation for anti-myeloma effects and for improvement of bone disease.

Deficiency of IL-17A, but not the prototypical Th17 transcription factor RORγt, decreases murine spontaneous intestinal tumorigenesis.

Abstract While inflammation has been associated with the development and progression of colorectal cancer, the exact role of the inflammatory Th17 pathway remains unclear. In this study, we aimed to determine the relative importance of IL-17A and the master regulator of the Th17 pathway, the transcription factor RORγt, in the sporadic intestinal neoplasia of APCMIN/+ mice and in human colorectal cancer. We show that levels of IL-17A are increased in human colon cancer as compared to adjacent uninvolved colon. Similarly, naïve helper T cells from colorectal cancer patients are more inducible into the Th17 pathway. Furthermore, IL-17A, IL-21, IL-22, and IL-23 are all demonstrated to be directly mitogenic to human colorectal cancer cell lines. Nevertheless, deficiency of IL-17A but not RORγt is associated with decreased spontaneous intestinal tumorigenesis in the APCMIN/+ mouse model, despite the fact that helper T cells from RORγt-deficient APCMIN/+ mice do not secrete IL-17A when subjected to Th17-polarizing conditions and that Il17a expression is decreased in the intestine of RORγt-deficient APCMIN/+ mice. Differential expression of Th17-associated cytokines between IL-17A-deficient and RORγt-deficient APCMIN/+ mice may explain the difference in adenoma development.

T1155 Anticancer Activity of a Broccoli Derivative, Sulforaphane, in Barrett's Adenocarcinoma: Potential use in Chemoprevention and as Adjuvant in Chemotherapy

INTRODUCTION The incidence of Barrett esophageal adenocarcinoma (BEAC) has been increasing at an alarming rate in western countries. In this study, we have evaluated the therapeutic potential of sulforaphane (SFN), an antioxidant derived from broccoli, in BEAC. METHODS BEAC cells were treated with SFN, alone or in combination with chemotherapeutic, paclitaxel, or telomerase-inhibiting agents (MST-312, GRN163L), and live cell number determined at various time points. The effect on drug resistance/chemosensitivity was evaluated by rhodamine efflux assay. Apoptosis was detected by annexin V labeling and Western blot analysis of poly(ADP-ribose) polymerase cleavage. Effects on genes implicated in cell cycle and apoptosis were determined by Western blot analyses. To evaluate the efficacy in vivo, BEAC cells were injected subcutaneously in severe combined immunodeficient mice, and after the appearance of palpable tumors, mice were treated with SFN. RESULTS SFN induced both time- and dose-dependent decline in cell survival, cell cycle arrest, and apoptosis. The treatment with SFN also suppressed the expression of multidrug resistance protein, reduced drug efflux, and increased anticancer activity of other antiproliferative agents including paclitaxel. A significant reduction in tumor volume was also observed by SFN in a subcutaneous tumor model of BEAC. Anticancer activity could be attributed to the induction of caspase 8 and p21 and down-regulation of hsp90, a molecular chaperon required for activity of several proliferation-associated proteins. CONCLUSIONS These data indicate that a natural product with antioxidant properties from broccoli has great potential to be used in chemoprevention and treatment of BEAC.

Abstract 1978: The poly-SUMO protein specific E3 ubiquitin ligase RNF4 is induced in multiple myeloma and reduces bortezomib-induced cell killing

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Multiple Myeloma (MM) is a fatal neoplasm of B-cell origin characterized by the clonal proliferation and accumulation of malignant plasma cells in the bone marrow. While recent advances in mechanistic understanding and treatment modalities have extended median survival to >6 years and 10% of patients survive >10 years, the vast majority of MM remains incurable with conventional, high dose therapy or stem cell transplantation. Bortezomib is a selective, reversible inhibitor of the 26S proteasome that inhibits protein degradation and is now FDA-approved for the treatment of newly diagnosed, relapsed and refractory MM. Though the catabolism of ubiquitinated substrates has been targeted therapeutically with significantly improved prognosis, patient response to bortezomib remains highly variable and cannot be predicted accurately. E3 ligases confer specificity on target selection for Ub+proteasome degradation. We therefore analyzed the expression of individual E3s using a microarray dataset obtained from MM patient tumor samples and found a striking variability in the expression level of individual E3 ligases between normal plasma cells and patients MM cells. RNF4, an E3 specific for poly-sumoylated proteins, was induced in MM patients and correlated with decreased patient response to the proteasome inhibitor bortezomib. Expression profiling of pretreatment tumor samples obtained from MM patients in independent clinical trials were used to generate a signature that correlated expression of SUMO+Ub+Proteasome pathway components with clinical outcome to predict patient response to bortezomib. Experimental validation by overexpression of RNF4-wt rendered myeloma cell lines relatively resistant to bortezomib while RNF4 depletion by shRNA enhanced sensitivity to bortezomib. Transfection of HA-tagged SUMO followed by bortezomib exposure led to the accumulation of HA-SUMO∼conjugates that were immunoreactive with Ub and proteasome components to further link the pathways. In summary, RNF4 an E3 ligase specific for SUMO-conjugates and induced in myeloma, modulates the cellular response to proteasome-based therapy and promotes bortezomib resistance. Our results support regulators of the sumoylation pathway as biomarkers to predict clinical response to bortezomib and provide evidence for targeting SUMO pathway to improve therapeutic outcome in myeloma in general and bortezomib specifically. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1978.