Abstract Constitutive activation of the NF-κB pathway is required for survival of the activated B cell–like (ABC) subgroup of diffuse large B-cell lymphoma (DLBCL). Here we show that a small molecule IκB kinase (IKK) inhibitor, PS-1145, and related compounds are toxic for ABC DLBCL cell lines but not for cell lines derived from the other prevalent form of DLBCL, germinal center B cell–like DLBCL. Treatment of ABC lines with these inhibitors rapidly induced a series of gene expression changes that were attributable to cessation of constitutive IKK activity, similar to changes induced by acute expression of genetic inhibitors of NF-κB, confirming the effectiveness and specificity of this compound. Before cell death, inhibition of IKK also induced features of apoptosis and an arrest in the G1 phase of the cell cycle. To test further the specificity of this toxicity, an inducible form of NF-κB was created by fusing the p65 NF-κB subunit with the ligand-binding domain of the estrogen receptor (p65-ERD). In the presence of tamoxifen, p65-ERD reversed the toxicity of IKK inhibition and restored expression of many NF-κB target genes. Another subgroup of DLBCL, primary mediastinal B-cell lymphoma (PMBL), also expresses NF-κB target genes, and treatment of a PMBL cell line with an IKK inhibitor was toxic and induced gene expression changes of a distinct group of NF-κB target genes. These studies validate the NF-κB pathway as a promising therapeutic target in ABC DLBCL, PMBL, and other lymphomas that depend on the activity of NF-κB for survival and proliferation.
Abstract Recently, T cell based immunotherapies have moved to the forefront of cancer immunotherapy with the success of Adoptive T cell therapy (ACT) and Immune checkpoint blockade. ACT, where patients are treated with tumor infiltrating T cells (TILs), conferred a clinical response rate of ∼50%. Treatment with anti-CTLA4 therapy, Ipilimumab, conferred response rates of 10-20%, greatly improving the overall survival of patients with advanced melanoma. Despite the encouraging outcomes, there are relatively low response rates coupled with the delay of weeks to months before tumor shrinkage can be appreciated. Thus, understanding mechanisms of resistance to immune therapies, to improve response rates, shorten time to treatment effect and developing predictive biomarkers of response are vital to the care of melanoma patients. In order to identify possible resistance mechanisms to immunotherapy, a high-throughput in vitro screen with 850 different bio-active compounds (Selleckchem), was designed to search for agents that could either increase or decrease the resistance of melanoma tumor cells to T cell mediated killing. Paired patient derived human melanoma tumor samples and TILs were used to assess which compounds when used to treat the melanoma cell lines can enhance the cytotoxic activity of the TILs against the paired melanoma sample, using a flow cytometry based assay in which active caspase 3 was used as a read out of apoptosis. We identified heat shock protein 90 (HSP90) inhibitors amongst the top compounds that improved T cell mediated cytotoxicity of treated tumor cells. We show that treatment with the HSP90 inhibitor ganetespib (Synta) greatly improves T cell mediated cytotoxicity of human cancer cells lines in vitro. Furthermore, in vivo murine studies using the MC38/gp100 tumor model show that ganestespib in combination with anti-CTLA4, resulted in superior antitumor effect and survival compared to either treatment alone (Average tumor volume at day 21 of treatment: Vehicle 294.3mm3, α-CTLA4 193 mm3, Ganetespib 237.5 mm3 and Ganetespib + α-CTLA4 105.8 mm3, P < 0.0001). Microarray analysis of human cell lines treated with ganetespib in vitro revealed an increase in interferon response genes including IFIT1, IFIT2, IFIT3. We confirmed these findings with quantitative real time PCR and western blot analyses and found IFIT1, IFIT2 and IFIT3 to be consistently upregulated across multiple melanoma cell lines following treatment with ganetespib. We next sought to verify the importance of the IFIT genes in the synergy observed between ganetespib treatment and T cell killing. First, we overexpressed IFIT1, IFIT2 and IFIT3 in human melanoma cell lines to recapitulate the improved sensitivity of the human melanoma cell lines to T cell killing following treatment with ganetespib. We then co-cultured these cells with their autologous T cells and found that overexpressing IFIT1, IFIT2 and IFIT3 mimicked the effects of ganetespib by increasing the sensitivity to T cell killing over the GFP control. On the other hand, silencing IFIT1, IFIT2 and IFIT3 simultaneously, abrogated the synergy between ganetespib and T cell killing. We are further elucidating the role of these genes in lowering the apoptotic threshold of cancer cells and contributing to the synergy of ganetespib and immunotherapy. This will enable the emergence of a new combination therapy of HSP90 inhibitors and anti-CTLA4 for the treatment of melanoma patients that will increase the percentage of patients responding to immunotherapy and achieving long term responses. Citation Format: Rina M. Mbofung, Jodi A. McKenzie, Shruti Malu, Chengwen Liu, Weiyi Peng, Isere Kuiatse, Leila Williams, Seram Devi, Zhe Wang, Trang Tieu, Tim Heffernan, Richard E. Davis, Rodabe Amaria, Patrick Hwu. HSP90 inhibitor, ganetespib, enhances responses to cancer immunotherapy through increased expression of interferon response genes [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B105.
TPS7575 Background: Diffuse large B-cell Lymphoma (DLBCL), the most common lymphoid malignancy, is categorized by the putative cell of origin (COO) classification system into germinal center (GCB) and non-GCB subtypes. Both the immunomodulatory agent lenalidomide (L) and BTK inhibitor ibrutinib (I) have shown activity in the non-GCB DLBCL subtype as single agents and in combination with chemotherapy. Separate randomized phase III trials of RCHOP alone and +L or +I are ongoing in non-GCB DLBCL. Preclinical studies demonstrate L+I results in synthetic lethality in non-GCB DLBCL via interferon signaling (Yang et al, Cancer Cell, 2012), but no trials have evaluated the efficacy in newly diagnosed patients. We present the first "window of opportunity" trial to use targeted therapy (rituximab, R+L+I) alone prior to chemotherapy in newly diagnosed DLBCL. Methods: In this investigator initiated phase II trial, patients receive standard R dosing q 21 days (d), L 25mg po d1-10, I 560mg po d1-21, and after two 21d RLI cycles or earlier if disease progression will start RLI-EPOCH q 21d for 6 cycles, without maintenance therapy. EPOCH will be dose adjusted based on standard criteria, capped at level +2. L+I dosing may be reduced if significant toxicity criteria are met. Key eligibility criteria include histologically confirmed, treatment-naïve non-GCB DLBCL, age ≥ 18, adequate performance status and organ function, and measurable disease. COO will be determined via immunohistochemistry for eligibility, and NanoString (Scott et al, Blood. 2014) for confirmation. Patients will be restaged with PET/CT using Lugano criteria (Cheson et al, JCO 2014). The primary objectives are to determine the overall response rate after two RLI cycles and complete response rate after 6 RLI+EPOCH cycles. Secondary objectives include safety and survival outcomes. Exploratory objectives include evaluation of baseline and therapy induced changes in gene and protein expression, mutations, minimal residual disease monitoring, and immune cell subsets in comparison with clinical outcomes. The trial will accrue 60 patients, and employs Bayesian futility and toxicity monitoring rules. Clinical trial information: NCT02636322.
Abstract Background Follicular lymphoma (FL) and marginal zone lymphoma (MZL) are indolent non‐Hodgkin lymphomas (iNHL). Median survival for iNHL is approximately 20 years. Because standard treatments are not curative, patients often receive multiple lines of therapy with associated toxicity—rationally designed, combination therapies with curative potential are needed. The immunomodulatory drug lenalidomide was evaluated in combination with rituximab for the frontline treatment of FL in the phase 3 RELEVANCE study. Ibrutinib, an oral Bruton tyrosine kinase inhibitor, is active in NHL and was evaluated in combination with lenalidomide, rituximab, and ibrutinib (IRR) in a phase 1 study. Methods The authors conducted an open‐label, phase 2 clinical trial of IRR for previously untreated FL and MZL. The primary end point was progression‐free survival (PFS) at 24 months. Results This study included 48 participants with previously untreated FL grade 1–3a ( N = 38), or MZL ( N = 10). Participants received 12, 28‐day cycles of lenalidomide (15 mg, days 1–21 cycle 1; 20 mg, cycles 2–12), rituximab (375 mg/m 2 weekly in cycle 1; day 1 cycles 2‐12), and ibrutinib 560 mg daily. With a median follow‐up of 65.3 months, the estimated PFS at 24 months was 78.8% (95% confidence interval [CI], 68.0%–91.4%) and 60‐month PFS was 59.7% (95% CI, 46.6%–76.4%). One death occurred unrelated to disease progression. Grade 3–4 adverse events were observed in 64.6%, including 50% with grade 3–4 rash. Conclusions IRR is highly active as frontline therapy for FL and MZL. Compared to historical results with lenalidomide and rituximab, PFS is similar with higher grade 3–4 toxicity, particularly rash. The study was registered with ClinicalTrials.gov (NCT02532257).
