Moon‐Tasson, Laurie L.

Abstract Abstract 1831 Poster Board I-857 IL-1 Antagonists Are More Effective at Inhibiting IL-6 Production than Apoptosis Inducing Agents: Implications for Targeting the Myeloma Proliferative Component. John A. Lust, MD,PhD1, Steven R. Zeldenrust, MD, PhD1, Laurie L. Moon-Tasson1*, and Kathleen A. Donovan, PhD1*. 1Division of Hematology, Mayo Clinic, Rochester, MN, United States, 55905. Background Multiple myeloma patients with an elevated growth rate have a shortened duration of response and survival. IL-6 is a central myeloma growth factor and we have shown that abnormal production of IL-1beta in the myeloma microenvironment stimulates the generation of paracrine IL-6 and myeloma cell growth in vivo. Dexamethasone and IL-1Ra both have the ability to inhibit IL-1 induced IL-6 production in vitro however their ability to inhibit IL-6 production and myeloma cell growth have not been investigated in a comparative fashion. Methods In vitro, IL-1 (100, 10, 1 pg/ml) was added to stromal cell cultures in the presence or absence of IL-1Ra (1 and 0.1 μg/ml) or dexamethasone (100 and 10 μM). IL-6 levels were quantitated by ELISA. In vivo, a patient with smoldering myeloma (≥ 10% bone marrow plasma cells) received 100 mg of IL-1Ra SQ qd for 6 months, low dose dexamethasone (20 mg qweek) for 6 months, followed by the combination of IL-1Ra and dexamethasone for 6 months. The bone marrow plasma cell percentage (BMPC), serum IgG, the plasma cell labeling index (marker of myeloma cell proliferation) and the C-reactive protein (marker of IL-6 production) were monitored serially. Results The effects of IL-1Ra and dexamethasone on IL-1 induced IL-6 production by marrow stromal cells are detailed in Figure 1. The results showed that IL-1Ra at 1 μg/ml was able to inhibit IL-1 induced IL-6 production back to baseline at all IL-1 concentrations tested. The inhibitory effect was less pronounced at 0.1 μg/ml IL-1Ra but still superior to dexamethasone. Dexamethasone was less effective at IL-6 inhibition compared to IL-1Ra using 100 and 10 pg/ml of IL-1 but similar at 1 pg/ml of IL-1. Of interest, the patient treated with IL-1Ra alone, dexamethasone alone, and the combination appeared to mimic these results in vivo. Anakinra alone induced a reduction of the PCLI and CRP. Dexamethasone alone decreased the M-protein; however the PCLI and CRP values increased. The PCLI increased from 0% to 0.8% and the CRP increased from 0.18 up to 1.48 indicating increased levels of IL-6 and a more active proliferative component of the disease. Subsequently, the combination of dexamethasone and IL-1Ra led to a further decrease in the IgG and the PCLI and CRP decreased again; PCLI decreased from 0.8% to 0.2% and CRP decreased from 1.48 down to 0.40. Conclusion The above results suggest that agents such as IL-1Ra are more effective at IL-6 inhibition and targeting the proliferative myeloma component than apoptosis inducing agents such as dexamethasone. Combination therapy with IL-1 inhibitors and apoptosis inducing agents may be useful in patients with active myeloma that have an elevated PCLI at diagnosis. Disclosures No relevant conflicts of interest to declare.

We report the long‐term follow‐up results of a phase II trial of IL‐1 receptor antagonist and low‐dose dexamethasone for early stage multiple myeloma (MM). Patients were eligible if they had smoldering multiple myeloma (SMM) or indolent multiple myeloma (IMM) without the need for immediate therapy. Forty seven patients were enrolled and subsequently treated with IL‐1Ra; in 25/47 low‐dose dexamethasone (20 mg weekly) was added. The primary endpoint was progression‐free survival (PFS). In the clinical trial, three patients achieved a minor response (MR) to IL‐1Ra alone; five patients a partial response (PR) and four patients an MR after addition of dexamethasone. Seven patients showed a decrease in the plasma cell labeling index (PCLI) which paralleled a decrease in the high sensitivity C‐reactive protein (hs‐CRP). The median PFS for the 47 patients was 1116 days (37.2 months). The median PFS for patients without (n = 22) and with (n = 25) a decrease in their baseline hs‐CRP was 326 days (11 months) vs. 3139 days (104 months) respectively (P <0.0001). The median overall survival (OS) for the 47 patients was 3482 days (9.5 years). The median OS for patients without and with a decrease in their baseline hs‐CRP was 2885 days (7.9 years) vs. median not reached, respectively (P = 0.001). In SMM/IMM patients at risk for progression to active myeloma, reduction in the hs‐CRP indicates successful targeting of the IL‐1/IL‐6 axis resulting in improved PFS and OS.(Clinical Trials.gov Identifier: NCT00635154) Am. J. Hematol. 91:571–574, 2016. © 2016 Wiley Periodicals, Inc.

Abstract Inflammation is a driving factor in the pathogenesis of many cancers including multiple myeloma. Myeloma is a plasma cell tumor whose early stages are characterized by cytokine dependent growth, predominantly driven by IL-6. We have demonstrated that IL-1β is a key inducer of IL-6 expressed by bone marrow stromal cells in a paracrine fashion. The P2X7 receptor (P2X7R) is an ATP-gated ion channel expressed by cells of the hematopoetic lineage that has been shown to play a critical role in IL-1β processing and secretion. We investigated the role this receptor may play in the IL-1/IL-6 cytokine pathway in multiple myeloma. A panel of multiple myeloma cell lines was evaluated by RT-PCR for the expression of P2X7R mRNA. Levels of expression in ANBL-6, MM1S, U266, RPMI-8226 and KAS-6/1 were comparable to U937, a previously characterized P2X7R positive cell line. A newly derived plasmacytoma cell line, PCYT3, showed very low levels of message expression. The ability of ATP to trigger the processing and release of IL-1β by activation of the P2X7 channel was studied using KAS-Pro, a myeloma cell line stably transfected with a pro- IL-1β gene. IL-1β, measured by ELISA, was released in a dose dependent fashion in response to increasing amounts of ATP ranging from 1mM to 5mM. Three P2X7R specific antagonists were tested for their effect on ATP stimulated IL- 1β release from KAS-Pro; all 3 compounds demonstrated inhibition in a dose dependent manner with varying degrees of potency correlating with P2X7R inhibition. In addition fresh patient samples (15 multiple myelomas/ 4 smoldering myelomas/8 MGUS) were evaluated for inhibition of IL-1β release by the P2X7R antagonists through direct measurement of IL-1β by ELISA as well as by an indirect measurement of IL-1β induced IL-6 production by normal bone marrow stromal cells. Greater than 90% inhibition of IL-1β release was demonstrated in all MM & SMM patients positive for IL-1β expression. IL-1β was not detected in any of the MGUS patients and therefore no affect of the inhibitors was observed. These results confirm the activity of functional P2X7 receptors on myeloma cell lines and in fresh patient samples and suggest a role for the use of P2X7 receptor antagonists in the therapy of myeloma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2582. doi:10.1158/1538-7445.AM2011-2582

Abstract Background: IL-6 is a central myeloma growth factor and in vitro abnormal production of IL-1beta in the myeloma microenvironment stimulates the generation of paracrine IL-6. Mimicking in vitro observations, we have shown that responsive early stage myeloma patients at risk for progression to active myeloma who were treated with IL-1 inhibitors demonstrated decreases in the myeloma proliferative rate and C-reactive protein (IL-6 surrogate marker) leading to a chronic disease state with an improved progression free survival. In this study we investigated the effect of XOMA 052, a humanized anti-IL-1 antibody to inhibit stromal cell IL-6 production induced by either recombinant IL-1beta or supernatants generated from unsorted bone marrow cells from patients with smoldering multiple myeloma. Methods: Bone marrow stromal cells were incubated with varying concentrations of recombinant IL-1β (100, 10, 1, 0.1 pg/ml) in the absence or presence of anti-IL-1 antibody, anti-TNF antibody, or dexamethasone. In a similar fashion, supernatants from unsorted bone marrow cells from six patients with smoldering myeloma were co-cultured with bone marrow stromal cells and the effect of the humanized anti-IL-1β antibody was tested. Results: The anti-IL-1β antibody inhibited the IL-1 induced IL-6 production in vitro by &gt; 85% at 100 pg/ml of IL-1 and &gt; 90% at 10, 1, and 0.1 pg/ml of IL-1; specifically, using 10 pg/ml of IL-1, IL-6 production was inhibited from 149 down to 5.2 ng/ml in the presence of anti-IL-1 antibody. Anti-TNF antibody had minimal effect. At 10 pg/ml of IL-1 beta, a physiologically relevant amount of IL-1 in vitro in bone marrow samples from myeloma patients, the anti-IL-1 antibody was superior to dexamethasone at inhibition of IL-6 production. In in vitro testing of patient supernatants from unsorted bone marrow cells, three patients were high inducers and three patients low inducers of paracrine IL-6 production. The results demonstrate &gt; 85% reduction of IL-6 production in all patients' samples tested. Importantly, the anti-IL-1β antibody was highly effective in samples from the three patients who were high inducers of paracrine IL-6 production (IL-6 production was inhibited from 191 down to 12 ng/ml; 157 to 13 ng/ml and 320 to 11 ng/ml). In comparison, the anti-TNF antibody had marginal effects. Conclusion: The humanized anti-IL-1beta antibody is highly effective in vitro at the inhibition of IL-6 generated by supernatant cultures from patients with early stage myeloma or by recombinant IL-1. Combination therapy with IL-1 inhibitors and apoptosis inducing agents may be useful in patients with active myeloma that have elevated IL-6 levels and a high growth rate at diagnosis. 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 2449.