Reversible Proteasome Inhibitor Research Articles

339 - Open-Label Study to Determine the Feasibility of Ixazomib as Maintenance after Allogeneic Stem Cell Transplant for Multiple Myeloma

Abstract Introduction: Allogeneic stem cell transplantation is an appropriate treatment option in patients (pts) with high-risk multiple myeloma (MM), but carries a mortality risk as a consequence of graft-versus-host disease (GVHD) and relapsed disease. Proteasome inhibitors have been shown to stimulate allogeneic immune cells to eliminate myeloma cells and at the same time reduce the incidence and severity of GVHD. The use of proteasome inhibitors after stem cell transplant can therefore prolong remissions after transplant and reduce the incidence of GVHD. Ixazomib is an orally bioavailable, reversible inhibitor of the 20S proteasome that has also shown impressive anti-myeloma activity in both the relapsed/refractory and the upfront setting. This is a Phase II, open-label, multicenter, non-randomized study to determine the feasibility of Ixazomib as maintenance after allogeneic stem cell transplant for MM. Here we present results from pts in the three treatment cohorts. Methods: High risk pts (including chromosome 17p, partial deletion (del(17p), t(4;14), t(14;16), t(14;20), plasma cell leukemia, or progression-free survival of less than 2 years after autologous stem cell transplant) who received an allogenic stem cell transplant and had no evidence of >Grade 2 GVHD or any GVHD requiring methylprednisolone were eligible to enroll between Days 45 and 120 after transplant. Pts received a weekly oral ixazomib dose on Days 1, 8, and 15 of each 28-day cycle for 6 cycles. Pts were treated at 2.3 mg (Cohort 1), 3 mg (Cohort 2), and 4 mg (Cohort 3). Results: To date, 7 pts were enrolled (3 on Cohort 1, 3 on Cohort 2, and 1 on Cohort 3); median age 49 years; 5 pts (71%) female; 4 pts (57%); International Staging System Grade ≥ 2. The median number of prior systemic regimens was 4 (range 3-8). 100% of pts had received autologous stem cell transplant. Median number of prior autologous transplants was 2 (range 1-3). Conditioning regimen prior to allogeneic stem cell transplant included fludarabine + melphalan (n = 6) and fludarabine + total body irradiation (n = 1). All pts received peripheral blood stem cells, donors included matched related donors (n=2) and unrelated donors (n=5). The median number of weeks of treatment were Cohort 1, 24 wks; Cohort 2, 16 wks; Cohort 3, 6 wks. To date, there have been no dose-limiting toxicities, 1 dose interruption on Cohort 1 due to thrombocytopenia, and 1 dose reduction on Cohort 2 due to rash. 5 pts (71%) have discontinued treatment, 3 pts (43%) due to disease progression and 2 pts (29%) have completed treatment. 2 pts (29%) were still on active treatment at the time of data cut-off. 3 pts (43%) have died due to disease progression. The median time on study overall was 9 months (range 2-28 months). Of the 6 pts (86%) eligible for response analysis, 2 pts (33%) had a stringent complete response, 1 pt (17%) had a complete response, 2 pts (33%) had a very good partial response, and 1 pt (17%) had progressive disease as a best overall response. The most common ≥ Grade 3 adverse events (AEs) were thrombocytopenia (3 pts, 43%) and hypertension (2 pts, 29%). 3 pts (43%) had ≥ Grade 3 treatment-related AEs of thrombocytopenia (3 pts, 43%) and rash (1 pts, 14%). 1 pt (14%) had treatment-related serious adverse events (vomiting and fever). Acute GVHD was reported for 5 pts (maximum Grade 1), chronic GVHD occurred in 4 pts (limited: 2 pts, extensive: 2 pts). Conclusions: There have been no dose-limiting toxicities on these 3 cohorts. The maximum-tolerated dose has not been reached for this study and enrollment is continuing. Initial response data is promising. Disclosures Matous: Celgene: Speakers Bureau; Multiple Myeloma Advisory Committee: Membership on an entity's Board of Directors or advisory committees. Berdeja: Vivolux: Research Funding; Constellation: Research Funding; Takeda: Research Funding; Curis: Research Funding; Teva: Research Funding; Novartis: Research Funding; Janssen: Research Funding; Celgene: Research Funding; BMS: Research Funding; Bluebird: Research Funding; Amgen: Research Funding; Abbvie: Research Funding.

Ubiquitin-proteasome system modulates zygotic genome activation in early mouse embryos and influences full-term development.

Maternal RNA/protein degradation and zygotic genome activation (ZGA), occurring during maternal-to-zygotic transition (MZT), are the first essential events for the development of pre-implantation embryos. Previously, we have shown the importance of the ubiquitin-proteasome system (UPS) for initiation of minor ZGA at the 1-cell stage of mouse embryos. However, little is known about the mechanism of involvement of the UPS-degraded maternal proteins in ZGA. In this study, we investigated the effect of inhibiting maternal protein degradation by the reversible proteasome inhibitor, MG132, on post-implantation development and ZGA regulation during early cleavage stages. Our study revealed that zygotic transcription by RNA polymerase II (Pol II) at the 1-cell stage was delayed and the full-term development was affected by transient proteasome inhibition during 1 to 9 h post-insemination (hpi). Furthermore, we found that the transient inhibition of proteasome activity at the 2-cell stage delayed the onset of transcription of some major ZGA genes. These results support the model hypothesizing the requirement of sequential degradation of maternal proteins by UPS for the proper onset of ZGA and normal progression of MZT in early mouse embryos.

Abstract 2051: LSTRA cell line as a model for large granular lymphocytic leukemia in drug screening

Abstract Large granular lymphocytic (LGL) leukemia is a rare form of leukemia that is caused by the increase in cytotoxic T-lymphocytes or natural killer (NK) cells. A subcategory of T-LGL leukemia that co-expresses T and NK cell surface markers is very aggressive and has a very poor prognosis. Current treatment of aggressive T-LGL is based on non-specific immunosuppressive therapy. Lack of model cell lines contributes to slow progress in designing targeted therapy and clinical trials. Our recent studies demonstrate that the mouse T-leukemic cell line LSTRA reproduces some important characteristics of aggressive T-LGL leukemia. This is the first report of a cell line model mimicking the deadly human leukemia and has great potentials for drug screening in treating T-LGL leukemia. Bortezomib is one of the drugs that show promising results in treating LGL leukemia patients in clinical trials. Bortezomib is the first FDA-approved reversible proteasome inhibitor in treating multiple myeloma and mantle cell lymphoma patients. One of the major concerns in using single-agent bortezomib is that patients develop resistance during the course of treatment and cancers relapse. Therefore, it is important to develop novel strategies in overcoming LGL leukemic cells’ resistance to bortezomib and its derivatives. In our current study, we showed that bortezomib inhibited cell proliferation and induced apoptosis in LSTRA leukemic cells. Partial reversion after 48-72 hr of bortezomib treatment suggested development of resistance to bortezomib. We further established bortezomib-resistant LSTRA cells that tolerated significantly higher levels of bortezomib. Both carfilzomib (FDA-approved second generation irreversible proteasome inhibitor) and MLN 4924 (proteasome pathway upstream inhibitor) failed to overcome bortezomib resistance. Only luteolin, a common flavonoid found in plants, can effectively overcome bortezomib resistance. Metabolic reprogramming has been linked to drug resistance in cancer cells. Indeed, We found that bortezomib-resistant LSTRA cells had smaller cell size as well as reduced rate of glucose uptake, lactate secretion, and mitochondrial respiration. These results suggest that reduced mitochondrial activity may be an important molecular marker for LSTRA cell’s resistance to bortezomib. All together, we identified luteolin as a potential small molecule in overcoming LGL leukemia’s resistance to proteasome inhibitor therapy. Altered mitochondrial activity and cell metabolism in bortezomib-resistant cells provide additional targets in developing novel combined chemotherapy for cancer patients. Citation Format: Fu-Shin Chueh, Fu-Yu Chueh, Chao-Lan Yu. LSTRA cell line as a model for large granular lymphocytic leukemia in drug screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2051. doi:10.1158/1538-7445.AM2017-2051

Homoharringtonine enhances bortezomib antimyeloma activity in myeloma cells adhesion to bone marrow stromal cells and in SCID mouse xenografts

Despite the great progress in the treatment, multiple myeloma (MM) still remains incurable. Bortezomib (BTZ), a reversible inhibitor of the 26S proteasome, is very effective against MM but unable to eradicate the MM cells in bone marrow niche eventually causing the disease relapse. Homoharringtonine (HHT) is a known anti-leukemia drug that inhibits MM both in vitro and in vivo. This study aimed to investigate whether HHT could potentiate the anti-tumor activity of BTZ in MM cells cocultured with bone marrow stromal cells and in vivo xenograft models. We found that coculture of myeloma cells with a human stroma cell line significantly decreased the sensitivity of myeloma cells to BTZ treatment. HHT inhibited the proliferation of MM cells and potentiated the anti-myeloma effects of BTZ by inhibition of both canonical and noncanonical NF-κB pathways. HHT also enhanced the anti-myeloma effect of BTZ in vivo xenograft models. Taken together, our data suggest that HHT can enhance the anti-myeloma activity of BTZ both in vitro and in vivo, which may represent a new clinical treatment in MM.

Bortezomib augments lymphocyte stimulatory cytokine signaling in the tumor microenvironment to sustain CD8+T cell antitumor function.

Tumor-induced immune tolerance poses a major challenge for therapeutic interventions aimed to manage cancer. We explored approaches to overcome T-cell suppression in murine breast and kidney adenocarcinomas, and lung fibrosarcoma expressing immunogenic antigens. We observed that treatment with a reversible proteasome inhibitor bortezomib (1 mg/kg body weight) in tumor-bearing mice significantly enhanced the expression of lymphocyte-stimulatory cytokines IL-2, IL-12, and IL-15. Notably, bortezomib administration reduced pulmonary nodules of mammary adenocarcinoma 4T1.2 expressing hemagglutinin (HA) model antigen (4T1HA) in mice. Neutralization of IL-12 and IL-15 cytokines with a regimen of blocking antibodies pre- and post-adoptive transfer of low-avidity HA518-526-specific CD8+T-cells following intravenous injection of 4T1HA cells increased the number of pulmonary tumor nodules. This neutralization effect was counteracted by the tumor metastasis-suppressing action of bortezomib treatments. In bortezomib-treated 4T1HA tumor-bearing mice, CD4+T-cells showed increased IL-2 production, CD11c+ dendritic cells showed increased IL-12 and IL-15 production, and HA-specific activated CD8+T-cells showed enhanced expression of IFNγ, granzyme-B and transcription factor eomesodermin. We also noted a trend of increased expression of IL-2, IL-12 and IL-15 receptors as well as increased phosphorylation of STAT5 in tumor-infiltrating CD8+T-cells following bortezomib treatment. Furthermore, bortezomib-treated CD8+T-cells showed increased phosphorylation of mitogen-activated protein kinase p38, and Akt, which was abrogated by phosphatidylinositide 3-kinase (PI3K) inhibitor. These data support the therapeutic potential of bortezomib in conjunction with other immunotherapies to augment the strength of convergent signals from CD8+T-cell signaling molecules including TCR, cytokine receptors and downstream PI3K/Akt/STAT5 pathways to sustain CD8+T-cell effector function in the tumor microenvironment.

A Phase II Study of Bortezomib-GIFOX (Gemcitabine, Ifosfamide, Oxaliplatin) in Patients with Newly Diagnosed Natural-Killer/T-Cell Lymphoma

Background:Natural-killer/T-cell lymphoma (NKTL) is a subtype of non-Hodgkin's lymphoma with poor response to conventional chemotherapy. Gene expression profiling of NKTL revealed overexpression of NF-kB. Bortezomib (B) is a potent and reversible proteasome inhibitor which has shown single agent activity in preclinical models of NKTL in vitro and in vivo. Further, drug testing in mouse NKTL xenograft suggest gemcitabine (G), oxaliplatin (ox) and ifosfamide (if) were effective in inducing tumor regression. We have also previously published a patient with relapsed NKTL that was successfully treated with GIFOX therapy. Hence we conducted a study to evaluate the clinical efficacy of B-GIFOX in patients with newly diagnosed NKTL.Methods:This was an open-labelled prospective phase II study approved by the institutional review board. Patients with histologically confirmed NKTL with stage IB or bulky(X) and stage II-IV disease were included. Patients with stage IB/IX or II were treated with 4 cycles of B-GIFOX followed by radiotherapy. Patients with advanced stage disease were treated with 6 cycles of B-GIFOX. Intravenous B was dosed at 1.3mg/m2 on days 1, 4, 8, and 11 every 21 days. Dose and schedule of G, If and Ox were 1000mg/m2 on day 1, 5g/m2 on day 2 and 85mg/m2 on day 2 respectively. The primary objective was to estimate the overall response rates (ORR) and the secondary objectives were to estimate the progression free survival (PFS), overall survival (OS) and toxicities of this regimen. PFS was calculated from the start of treatment to the date of progression or last follow-up. OS was calculated from the start of treatment to the date of death or last follow-up. Both median PFS and OS were estimated using Kaplan-meier curves.Results:There were 7 patients recruited into the study; 3 had stage II disease and 4 had stage IV disease. The median age was 50 years (range: 34-61) and 5 (71.4%) were male. Four patients had B symptoms and all but one patient had elevated LDH at diagnosis. Six patients completed their planned treatment and one patient had disease progression through the first cycle of chemotherapy. The ORR was 42.8%; one patient had a complete response, two had partial responses and 4 had progressive disease. The median PFS was 4.3months (95% confidence interval [CI] 4.0-4.6 months) and the median OS was 14.9months (95% CI 0.6-29.2 months). The following grade 1-2 toxicities were seen in at least 1 patient: anemia (7), thrombocytopenia (5), fever (3), ALT increase (3), nausea (2), vomiting (2), ALP increase (2), AST increase (1), alopecia (1), fatigue (1), and body ache (1). The following grade 3-4 toxicities were seen in at least 1 patient: thrombocytopenia (2), and tumor lysis syndrome (1).Conclusion:This study showed that B-GIFOX induced an ORR of 43% in patients with newly diagnosed NKTL however the median PFS was short at around 4months. DisclosuresNo relevant conflicts of interest to declare.

Highlights of This Issue

Yoo and colleagues investigated the impact of bortezomib, a peptide-based reversible proteasome inhibitor, combined with oncolytic herpes simplex virus-1 on glioblastoma cell death and sensitivity to NK cell immunotherapy in vitro and in vivo. The authors show that combination treatment induced inflammatory necroptotic cell death and the secretion of inflammatory cytokines into the tumor microenvironment. This resulted in enhanced NK cell activation and increased NK cell-mediated killing of infected tumor cells. These findings provide the rationale for combination therapy for glioblastoma.Lipid metabolism reprogramming is a novel characteristic of malignancies. Geng and colleagues reveal that glioblastoma (GBM) patients possessing elevated lipid droplets (LD), special organelles storing cholesterol esters in cells, presented poor survival, demonstrating that LD might serve as a novel diagnostic biomarker in GBM. Blocking cholesterol storage via inhibition of SOAT1, a key enzyme controlling cholesteryl esterification, suppressed GBM growth and prolonged survival in xenograft models via inhibition of SREBP-1-regulated de novo lipid synthesis. LDs/cholesterol esters uniquely formed in tumor tissues provide an ideal target to specifically inhibit GBM cells while sparing normal brain tissues.The identification of cancer stem-like cells (CSC) may be essential for targeting cancer therapy. Detection of purified CSC via cell surface markers in colorectal cancer (CRC) has limitations. The authors show that low proteasome activity cells (LPAC) have characteristics of CSC and may serve as a means to purify CSC. Furthermore, EP300-interacting inhibitor of differentiation 3 is enriched in LPAC and is significantly related to colorectal cancer treatment resistance and patient prognostication. The finding of LPAC as CSC has implications for development of novel targeted cancer therapies.Immune checkpoint inhibitors have recently revolutionized cancer immunotherapy. Despite preclinical data showing the KIR2D-specific checkpoint inhibition antibody IPH2101 bolsters NK cell antitumor immunity, no responses were observed when evaluated in patients with smoldering myeloma. Carlsten and colleagues report that IPH2101 infusions unexpectedly triggered a rapid reduction in KIR2D expression on NK cells via trogocytosis and that NK cells that lost KIR2D expression, or had KIR2D masked by the antibody, promptly lost function due to detuning in vivo. These findings provide the first mechanistic insights into events that may compromise the efficacy of checkpoint inhibitor antibodies aimed at inducing “missing-self” tumor targeting by NK cells in cancer patients.

Cancer cachexia-when proteasomal inhibition is not enough.

Cachexia is a life threatening syndrome associated with several diseases, such as end‐stage heart failure, end‐stage renal disease, chronic obstructive pulmonary disease, chronic inflammation (i.e. rheumatoid arthritis), acquired immune deficiency syndrome, and cancer.1, 2 Cachexia is found in 31–87% of cancer patients especially in advanced disease stages.3 It is characterized by progressive weight loss, metabolic alterations, fatigue, and persistent reduction of body cell mass in response to a malignant tumour.2, 4, 5, 6 The incidence of cachexia in cancer patients is dependent on the type and site of the tumour. While patients with non‐Hodgkin's lymphoma, breast cancer, and sarcomas show low incidences, rates up to 83% in pancreatic cancer patients, and over 85% in patients with gastric cancer have been found. Additionally, around 60% of small‐cell and non‐small‐cell lung cancer patients develop cachexia.7, 8, 9 Cancer cachexia affects the function of several organs such as muscle, adipose tissue, liver, brain, immune system, and heart, collectively decreasing patients' quality of life and worsening their prognosis. Therefore, cachexia must be considered as a true multi‐organ syndrome.10 Because cancer cachexia leads to a decrease in physical performance and quality of life,11 and is associated with poor survival (accounting for more than 20% of cancer deaths,7, 12, 13, 14, 15) it is of major clinical relevance. Even more so since cachectic patients show lower response rates to chemotherapy7 and a reduced tolerance to anticancer treatment.16 Despite its importance, weight loss in cancer patients is rarely recognized, assessed,17 or treated actively.18, 19 Thus, cancer cachexia represents an important underappreciated clinical syndrome.

Bortezomib for the prevention and treatment of graft-versus-host disease afterallogeneic hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation is the standard treatment for a variety of benign and malignant conditions. However, graft-versus-host disease (GvHD) continues to present a major barrier to the success and wide applicability of this procedure. Although current GvHD prevention and treatment regimens exclusively target Tcells, bortezomib, a reversible proteasome inhibitor, possesses unique immune regulatory activities that span a wide variety of cellular processes of T and dendritic cells essential for the development of GvHD. Herein, we review the current understanding of the effects of bortezomib invitro and in animal models and summarize the clinical data relevant to its use in the prevention and treatment of GvHD. We conclude with an outline of the remaining challenges and opportunities to optimize bortezomib's potential role in this setting.

Ixazomib: First Global Approval.

Ixazomib (Ninlaro(®)) is an orally bioavailable, reversible proteasome inhibitor developed by Millennium Pharmaceuticals, Inc. (now Takeda Oncology). Ixazomib acts by binding to and inhibiting the β5 subunit of the 20S proteasome. In November 2015, the US FDA approved ixazomib for use in combination with lenalidomide and dexamethasone for the treatment of patients with multiple myeloma who have received at least one prior therapy. Ixazomib is under regulatory review for this indication in the EU. PhaseIII development of ixazomib is underway worldwide for newly-diagnosed multiple myeloma (in patients who are not eligible for stem cell transplant, or as maintenance therapy) and for relapsed or refractory systemic light chain (AL) amyloidosis. Ixazomib is also under phase I-II development for the treatment of several other haematological and non-haematological malignancies, graft-versus-host disease and lupus nephritis. This article summarizes the milestones in the development of ixazomib leading to this first approval for multiple myeloma.

Descriptive Analysis of Bortezomib Use in Multiple Myeloma in Four Adult University Teaching Hospitals in Quebec, Canada

Background: Bortezomib, a reversible inhibitor of the 26S proteasome widely used in the treatment of multiple myeloma, is now being used in various other indications. Pharmacy directors gave the Therapeutic Drug Management Program (TDMP - www.pgtm.qc.ca) the mandate to evaluate bortezomib use in four University Hospitals in Quebec, Canada.Objectives: Describe bortezomib use for all indications in our hospitals and review its utilisation in the treatment of multiple myeloma.Methods: A review of pharmacy databases was performed to identify patients who receivedbortezomib between June1st 2012 and May 31st 2013. The pharmaceutical and medical records of every patient who received bortezomib were reviewed to assess the treatment, pathology and adverse events.Results: Two hundred and thirty two bortezomib regimens were administered to 227 different patients during the study period. Median age was 68. The most frequent indication (55%) was first-line treatment of multiple myeloma (n=128) followed by treatment of relapsed/refractory disease (31%) (n=73). Various other indications, including amyloidosis (n=17), lymphoplasmacytic lymphoma (n=12) and mantle cell lymphoma (n=2), represented 13% of the population.At the time of data analysis, 35% of patients were still treated with bortezomib, 25% had finished their planned treatment and 34% had discontinued treatment because of adverse events or disease progression. Fifteen patients (6%) died during the study period.Among the 45 patients eligible for autologous stem cell transplant (ASCT), the main regimen used was the association of bortezomib and dexamethasone (VelDex) (n=27), primarily using subcutaneous bortezomib (n=24) at 1.3 mg/m2 (n=30). Median treatment duration was four cycles. Twenty-eight patients have undergone ASCT and only two progressed.The association of bortezomib, melphalan and prednisone (VMP) (54.2%) followed by VelDex (29%) and the association of cyclophosphamide, bortezomib and dexamethasone (CyborD) (16.8%) were the regimens used in the population (n=83) not eligible to ASCT. Response rate using international uniform response criteria for multiple myeloma was 47.9% excluding patients still receiving treatment at the time of data collection.Seventy three patients received bortezomib for relapsed/refractory myeloma. Of these patients, thirty two (43.8%) discontinued therapy, nineteen due to disease progression, eight for the occurrence of side effects and five for other reasons. The initial dose was variable, from 1.0 to 1.6 mg/m2, and close to half of this patient population received CYBorD (49.4%), followed by VelDex (30.2%) and VMP protocols (15%). The number of cycles for patients who completed treatment (4 to 9) as well as the median exposure time (57 to 223 days) was also highly variable.Respectively 8.5% and 10.9% of the population treated with bortezomib for multiple myeloma were hospitalized (n=17) or had to discontinue treatment (n=22) because of adverse events (mostly hematologic toxicity, peripheral neuropathy or gastro-intestinal toxicity).Conclusions: Bortezomib is widely used in the treatment of multiple myeloma. Treatment algorithms should be developed and implemented to optimize the use of bortezomib, particularly in the relapsed/refractory setting. Standard regimens should also be implemented in each center. The utilisation of pre-printed orders for the prescription of chemotherapy regimens promotes uniform prescription. A review of the literature should be performed and recommendations should be made for the use of bortezomib in off-label indications like amyloidosis and lymphoplasmacytic lymphoma. DisclosuresOff Label Use: bortezomib use in amyloidosis and lymphoplasmacitic lymphoma. Lemieux-Blanchard:celgene: Membership on an entity's Board of Directors or advisory committees; Amgen and Janssen: Other: preceptorship. Bérard:Janssen: Honoraria. Sebag:Janssen: Honoraria; Amgen: Honoraria; Celgene: Honoraria; Novartis: Honoraria.

Preclinical Evaluation of the Oral Proteasome Inhibitor Ixazomib in Diffuse Large B-Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL), the most common human lymphoma, comprises a genetically and clinically diverse group of aggressive B cell non-Hodgkin lymphomas (NHL-B) among a small group of important human cancers increasing in incidence in the US over the last four decades.Current frontline DLBCL therapy although fairly successful (~70-80% remission rate with frontline R-CHOP chemo-immunotherapy), frequently relapses (~40% within 2-3yrs.) often with refractory (r/r) DLBCL leaving only very poor salvage therapy options (<20% PR/CR) and short survival. Of note, 2-3% of DLBCL patients with rearrangements of MYC and either BCL2 or BCL6 gene, so called double-hit lymphoma (DHL), are associated with an even more dismal outcome. Investigation of novel therapeutic approaches for refractory DLBCL is still in progress. Ixazomib (MLN9708), an investigational, orally bioavailable, small-molecule, reversible inhibitor of the 20S proteasome, was pre-clinically evaluated in refractory DLBCL models. We initially screened the activity of ixazomib in 28 DLBCL cell lines using viability assays with increasing doses of ixazomib (0-1000 nM) for 72 hours. Exposure of ixazomib resulted in a dose-dependent inhibition of cell viability with IC50s ranging from as low as 20 nM up to 200 nM. When we compared the IC50 values of ixazomib in DLBCL cell lines with other proteasome inhibitors such as bortezomib (BZ) and carfilzomib (CFZ), the IC50s of ixazomib (average 120 nM) is 16-fold higher than BZ (average 7.4 nM) and 8.8-fold higher than CFZ (average 13.5 nM). We have also evaluated the activity of ixazomib in primary cells from patients with refractory DLBCL (7 cases). The IC50 values of ixazomib in primary refractory DLBCL ranges from 40 nM to 200 nM. Similar drug concentrations of ixazomib were tested on normal peripheral blood mononuclear cells, but showed no significant cellular toxicity. The inhibition effect of ixazomib was noticeable in two representative DLBCL cell lines, MZ (GCB subtype) and RC (double hit-DLBCL) with IC50 well below 50 nM. Thus, we selected these cell line to test whether ixazomib can induce apoptosis. Annexin V assays showed that ixazomib effectively induces apoptosis in both MZ and RC cells in a dose- and time-dependent manner. To assess the functional significance of ixazomib-induced cell growth inhibition and apoptosis in DLBCL, proteomic approach by reverse phase protein array (RPPA) was used. RC and MZ cells were treated with ixazomib (IC75) for 12 hrs and 24 hrs, and protein extracts were analyzed by a customized RPPA with 285 validated antibodies. The profiles for RC and MZ after ixazomib treatment were quite different in that few common proteins get down/up-regulated. This could be due to the fact that RC is a double hit (BCL-2 x Myc) DLBCL cell line and utilizes different growth/survival pathways than MZ cells, particularly activation of protein synthesis pathway (IGF-PI3K-PDK1-ATK-mTOR-S6K-S6) and the growth/survival pathway (Fibronectin-FAK-MEK1-p38-ELK1). Ixazomib treatment significantly down-regulated these two pathways in RC cells. Several common proteins that were significantly up-regulated after 24 hrs of ixazomib treatment in both RC and MZ cells are the apoptotic related proteins (Caspase 3, cleaved caspase 7, and cleaved PARP) and the DNA damage related proteins (H2AX, Histone H-3, DM-Histone-H3, and HM-K9-Histone-H3). These results suggest that DNA damage could be one of ixazomib's mechanisms of action. In conclusion, our results indicate that ixazomib is an active proteasome inhibitor in refractory DLBCL cells, and warrants further investigation as a potential therapy for treatment of therapy-resistant DLBCL. DisclosuresPham:Amgen, Onyx, Millennium: Research Funding.

Phase 1 Study of Carfilzomib for the Prevention of Relapse and Graft-Versus-Host Disease in Allogeneic Hematopoietic Cell Transplantation for High-Risk Hematologic Malignancies

Allogeneic hematopoietic cell transplant (HCT) remains the primary curative option for patients (pts) with poor-risk hematologic malignancies, but graft-versus-host disease (GVHD) and relapse remain major obstacles. Of note, GVHD therapy with systemic steroid and more potent immunosuppression abrogates graft-versus-tumor effect and further increases relapse. The NF-κB signaling pathway has a pivotal role in maintaining malignant cell survival and in mediating inflammatory and immune response by promoting activated T and dendritic cell function and survival. Inhibition of proteasome function results in down-regulation of the NF-κB signaling pathway, thus selective apoptosis of malignant cells, in which this pathway is up-regulated, and of allo-reactive T and dendritic cells. The reversible proteasome inhibitor bortezomib may abrogate GVHD and relapse based on pre-clinical studies and a phase 1/2 clinical trial when added to high-risk mismatched unrelated donor HCTs for pts with hematologic malignancies. (Koreth J, et al. J Clin Oncol 2012;30(26):3202-8.) Carfilzomib is a second-generation irreversible immunoproteasome inhibitor, with potentially increased efficacy and specificity to the immune and hematologic malignancy cells. It has a less off-target toxicity profile, especially myelosuppression, neuropathy and diarrhea. We are exploring the addition of carfilzomib to fludarabine-based conditioning regimens and GVHD prophylaxis with tacrolimus and methotrexate.Primary Objective: To identify the maximal tolerated dose (MTD) of carfilzomib when administered on day +1, +2, +6, +7.Methods: The phase 1 design is standard 3+3. Eligible conditioning regimens include fludarabine/busulfan and fludarabine/melphalan. Tacrolimus is started on day -3 and tapered off by day +180 if ≥grade II acute GVHD do not occur. Methotrexate 5 mg/m2 is given IV on day +1, +3, +6, +11. Carfilzomib is administered IV on days +1, +2 at 20 mg/m2/day fixed dose and on day +6, +7 at 4 dose levels: 20, 27, 36, 45 mg/m2/day. Premedication with 4 mg dexamethasone IV is administered prior to each dose of carfilzomib 20 and 27 mg/m2 and with 8 mg prior to each dose of 36 and 45 mg/m2, respectively. Carfilzomib is given after IV methotrexate on day +1 and +6. Subcutaneous filgrastim 5 μg/kg/day is started on day +1 until engraftment. Dose-limiting toxicities (DLTs) are defined as any ≥grade 3 non-hematologic National Cancer Institute Common Terminology Criteria for Adverse Events version 4 toxicities which occur within 28 days of carfilzomib treatment (day +35) and are directly attributed to carfilzomib.Results: Since October 2014, 10 pts (Male: Female, 5:5) have been enrolled. Nine pts received all planned 4 doses and 1 female received 3 doses only before being removed due to unrelated infectious adverse events. Among 9 evaluable pts, the median age was 55 (range, 29-61) years; all donors were 8/8 matched (7 sibling, 2 unrelated). Stem cell sources were peripheral blood in 8 and bone marrow in 1 unrelated donor. Primary diseases included FLT3-ITD+ acute myeloid leukemia in 2, acute bi-phenotypic leukemia in 1, BCR/ABL+ acute lymphoblastic leukemia in 1, myelodysplastic syndrome in 2, lymphoma in 2, and multiple myeloma in 1. All pts received the myeloablative reduced-toxicity fludarabine and busulfan x 4 (FluBu4), with busulfan kinetics targeting the concentration at steady state at 600-900 ng/mL. One pt with diffuse large B cell lymphoma received rituximab-FluBu4. All 9 pts engrafted neutrophil and platelet at a median time of 10 (range, 10-15) and 12 (9-14) days, respectively. A pt of dose level 2 (20/27 mg/m2) who received a bone marrow stem cell dose of 1.3 x 106 CD34+ cells/kg unexpectedly engrafted as early as day +15. Expected common toxicities related to the FluBu4 treatment was oral mucositis. Median CD33+ and CD3+ donor chimerisms were 100% and 88% at day 30, respectively, and were 100% and 91% at day 100, respectively. As of 8/4/15, no DLTs occurred in the 8 pts who had passed day +35, with the median transplant day of day +172 (range, 65-292). The 9th pt of dose level 3 (20/36 mg/m2) is day +14 and has engrafted.Conclusion: Adding the irreversible immunoproteasome inhibitor carfilzomib to fludarabine-based conditioning HCT appears safe and feasible. Dose escalation to level 4 (20/45 mg/m2) is ongoing and the results will be updated at the meeting. DisclosuresPawarode:Onyx Pharmaceuticals: Research Funding. Off Label Use: Carfilzomib is an irreversible proteasome inhibitor and is FDA approved for treament of relapsed refractory multiple myeloma. It is here being investigated for its immunomodulatory and anti-tumor properties in the prevention of graft-versus-host disease (GVHD) and disease relapse when incorporated into fludarabine based conditioning and GVHD prophylaxis regimens for allogeneic hematopoietic cell transplantation in patients with high-risk hematologic malignancy.. Levine:Novartis: Consultancy.

Bortezomib in mantle cell lymphoma: comparative therapeutic outcomes.

Mantle cell lymphoma (MCL) is an incurable, typically aggressive subtype of non-Hodgkin lymphoma, accounting for 4%–7% of newly diagnosed non-Hodgkin lymphoma cases. Chemoresistance commonly ensues in MCL, and patients with this heterogeneous disease invariably relapse, underscoring the unmet need for better therapies. Over the past few years, several novel agents with promising activity and unique mechanisms of action have been deemed effective in MCL. Bortezomib is a reversible proteasome inhibitor, approved as a single agent for patients with relapsed/refractory MCL who have received at least one prior line of therapy. Addition of bortezomib to chemoimmunotherapies has demonstrated good tolerability and superior efficacy, both in the upfront and salvage settings, and recently one such combination of bortezomib plus rituximab, cyclophosphamide, doxorubicin, and prednisone was approved as a frontline regimen in untreated patients with MCL. This review examines the role of bortezomib in a multitude of clinical settings and ongoing clinical trials designed to optimize its integration in the current treatment paradigms of MCL.

Small ubiquitin-related modifier 2/3 interacts with p65 and stabilizes it in the cytoplasm in HBV-associated hepatocellular carcinoma.

BackgroundSUMOylation, an important post-translational modification, associates with the development of hepatocellular carcinoma (HCC). p65, one of the most important subunits of NF-κB, is a key regulator in the development of HCC and has been reported to be SUMOylated by exogenous small ubiquitin-related modifier 3 (SUMO3) in HEK 293T cells. However, the relationship between p65 and SUMO2/3 in HCC remains unknown. This study was to investigate the interaction between p65 and SUMO2/3 and explore the potential roles involved in HCC.MethodsThe expressions of p65 and SUMO2/3 in the liver tissues were detected by using immunohistochemistry. We performed double-labeled immunofluorescence and co-immunoprecipitation assay to verify the interaction between p65 and SUMO2/3. The extraction of nuclear and cytoplasmic proteins was performed, and the subcellular localization of p65 was detected. The proliferation and migration of hepatoma cells were observed using MTT, colony formation, and transwell assays.ResultsWe found a strong SUMO2/3-positive immunoreactivity in the cytoplasm in the non-tumor tissues of HCC. However, SUMO2/3 level was down regulated in the tumor tissues as compared with the adjacent non-tumor tissues. In accordance with this finding, p65 was up regulated in the adjacent non-tumor tissues and almost localized in the cytoplasm. There was a close correlation between SUMO2/3 and p65 expressions in the liver tissues (R = 0.800, p = 0.006). The interaction between p65 and SUMO2/3 was verified by co-immunoprecipitation and double-labeled immunofluorescent assays. TNF-α (10 ng/ml) treatment for 30 min not only up regulated the cytoplasmic conjugated SUMO2/3, but also enhanced SUMO2/3-p65 interaction. Furthermore, we found that SUMO2/3 up regulated the cytoplasmic p65 protein level in a dose-dependent manner, but not affected its mRNA level. The increase of p65 protein by SUMO2/3 was abolished by MG132 treatment, a reversible inhibitor of proteasome. Meanwhile, TNF-α-induced increase of SUMO2/3-conjugated p65 was along with the reduction of the ubiquitin-conjugated p65. The further study showed that SUMO2/3 over-expression decreased the proliferative ability of hepatoma cells, but did not affect the migration.ConclusionSUMO2/3-p65 interaction may be a novel mechanism involved in the transformation from chronic hepatitis B to HCC via stabilizing cytoplasmic p65, which might shed light on understanding the tumorigenesis and development.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1665-3) contains supplementary material, which is available to authorized users.

Physiologically-based pharmacokinetic modeling of target-mediated drug disposition of bortezomib in mice.

Bortezomib is a reversible proteasome inhibitor with potent antineoplastic activity that exhibits dose- and time-dependent pharmacokinetics (PK). Proteasome-mediated bortezomib disposition is proposed as the primary source of its nonlinear and apparent nonstationary PK behavior. Single intravenous (IV) doses of bortezomib (0.25 and 1 mg/kg) were administrated to BALB/c mice, with blood and tissue samples obtained over 144 h, which were analyzed by LC/MS/MS. A physiologically based pharmacokinetic (PBPK) model incorporating tissue drug-target binding was developed to test the hypothesis of proteasome-mediated bortezomib disposition. The final model reasonably captured bortezomib plasma and tissue PK profiles, and parameters were estimated with good precision. The rank-order of model estimated tissue target density correlated well with experimentally measured proteasome concentrations reported in the literature, supporting the hypothesis that binding to proteasome influences bortezomib disposition. The PBPK model was further scaled-up to humans to assess the similarity of bortezomib disposition among species. Human plasma bortezomib PK profiles following multiple IV dosing (1.3 mg/m(2)) on days 1, 4, 8, and 11 were simulated by appropriately scaling estimated mouse parameters. Simulated and observed bortezomib concentrations after multiple dosing were in good agreement, suggesting target-mediated bortezomib disposition is likely for both mice and humans. Furthermore, the model predicts that renal impairment should exert minimal influence on bortezomib exposure in humans, confirming that bortezomib dose adjustment is not necessary for patients with renal impairment.

Bortezomib in the Treatment of Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is a hematological malignancy with unfavorable prognosis. Bortezomib, a potent, selective and reversible inhibitor of the 26S proteasome, was shown to be active in MCL and is currently implemented in therapeutic combinations. Single-agent bortezomib has demonstrated clinical efficacy in relapsed and refractory MCL with objective response in up to 47% of the patients. However, complete remission rates are low and duration of response is relatively short. In previously untreated patients, the addition of bortezomib to induction chemotherapy is also promising. Further evaluation of bortezomib alone or in combination with other drugs for the treatment of MCL is warranted to improve the quality of life and survival of patients. This review explores bortezomib as therapy in patients with MCL.

MG132 enhances the radiosensitivity of lung cancer cells in vitro and in vivo.

Radiotherapy is a common treatment modality for lung cancer, however, radioresistance remains a fundamental barrier to attaining the maximal efficacy. Cancer cells take advantage of the ubiquitin-proteasome system (UPS) for increased proliferation and decreased apoptotic cell death. MG132 (carbobenzoxyl-leucinyl-leucinyl-leucinal‑H), a specific and selective reversible inhibitor of the 26S proteasome, has shown anticancer effect in multiple types of cancers. Previously, we have reported that MG132 enhances the anti‑growth and anti-metastatic effects of irradiation in lung cancer cells. However, whether MG132 can enhance the radiosensitivity in lung cancer cells in vitro and in vivo is still unknown. In this study, we found that MG132 increased apoptosis and dicentric chromosome ratio of A549 and H1299 cells treated by irradiation. Radiation-induced NF-κB expression and IκBα phosphorylation was attenuated in MG132 plus irradiation-treated cells. The in vivo model of H1299 xenografts of nude mice showed that the tumor size of MG132 plus irradiation treated xenografts was smaller than that of irradiation, MG132 or the control group. Moreover, MG132 plus irradiation group showed significant reduced Ki67 expression. Taken together, these results demonstrate that MG132 enhances the radiosensitivity through multiple mechanisms in vitro and in vivo.

Abstract 2702: Therapeutic potentials of STAT5 inhibitors in overcoming bortezomib resistance in human T-cell leukemia

Abstract The purpose of our study is to define the mechanism of bortezomib resistance in cancer therapy and to identify novel approaches in overcoming the resistance. Bortezomib is the first FDA-approved reversible proteasome inhibitor in treating multiple myeloma and mantle cell lymphoma patients. Numerous clinical trials are ongoing in testing the efficacy of bortezomib and its derivatives in treating human cancers. One of the major concerns in using single-agent bortezomib is that patients develop resistance during the course of treatment and cancers relapse. To explore the underlying mechanisms, we recently established a bortezomib-resistant human acute T lymphoblastic cell line Jurkat-BR. Jurkat-BR remains sensitive to carfilzomib, a FDA-approved second-generation irreversible proteasome inhibitor. An inhibitor targeting upstream proteasome pathway, MLN 4924, also exhibits cytotoxic effects toward Jurkat-BR. Overexpression and hyperactivation of signal transducer and activator of transcription 5 (STAT5) is associated with many forms of blood malignancies. Compared with parental Jurkat, Jurkat-BR has significant elevation of STAT5b, but not STAT5a, at both mRNA and protein levels. Pimozide is an antipsychotic drug that recently found to specifically inhibit STAT5 activity. Consistent with STAT5b overexpression, Jurkat-BR is more sensitive than parental Jurkat to cytotoxicity induced by pimozide. Another inhibitor targeting STAT5 SH2 domain, the nonpeptidic nicotinoyl hydrazone (CAS# 285968-31-4, STAT5I) also exhibits similar effects. Both pimozide and STAT5I synergize with bortezomib in killing Jurkat-BR cells. These results support the importance of STAT5b upregulation in acquired bortezomib resistance. We reported previously that STAT5 translocates to mitochondria (BBRC 402:778) and represses mitochondrial gene expression (manuscript submitted). In Jurkat-BR cells, we also observed STAT5b mitochondrial translocation, reduced mitochondrial gene expression, and repression of mitochondrial respiration. These results suggest that both STAT5b overexpression and reduced mitochondrial activity may be important molecular markers for cancer cell's resistance to bortezomib. Our data further support the clinical benefit of STAT5 inhibitors in complementing proteasome inhibitors and in overcoming its resistance in patients who acquire resistance through upregulating STAT5. More importantly, the link between reduced mitochondrial respiration and bortezomib resistance highlights the potentials of targeting mitochondria in developing novel combined chemotherapy for cancer patients. Citation Format: Fu-Yu Chueh, Shahrooz Vahedi, Fu-Shin Chueh, Chao-Lan Yu. Therapeutic potentials of STAT5 inhibitors in overcoming bortezomib resistance in human T-cell leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2702. doi:10.1158/1538-7445.AM2015-2702

Abstract 1768: Chloroquine potentiates carfilzomib but not bortezomib effects on myeloma cells

Abstract Proteasomal inhibition has proven good clinical effect in Multiple Myeloma; however, some patients are resistant to treatment and most patients acquire resistance during the disease course. It is well known that cells degrade proteins by two distinct but interconnected pathways; one is protein degradation by proteasomes, which may be clinically targeted by proteasome inhibitors, the other is by formation of autophagosomes and lysosomal degradation, which can be inhibited by hydroxychloroquine (HCQ). Assuming that the two arms of protein degradation may compensate for each other, there is a rationale to simultaneously target both arms. We tested the combined effects of HCQ and the reversible proteasome inhibitor bortezomib or the irreversible inhibitor carfilzomib on myeloma cell lines and primary cells. As expected, HCQ potentiated carfilzomib-induced myeloma cell death, but surprisingly, HCQ had little or no effect on bortezomib activity. We also found that HCQ potentiated the effect of the irreversible proteasome inhibitor oprozomib, but not the effect of the reversible inhibitor ixazomib. Thus, the sensitivity towards the irreversible proteasome inhibitors carfilzomib or oprozomib is to a larger extent affected by the autophagic system than the reversible inhibitors bortezomib and ixazomib. In conclusion, we here suggest that in attempts to increase the clinical efficacy of proteasome inhibitors, inhibitors of autophagy and lysosomal degradation such as HCQ should be combined with an irreversible proteasome inhibitor such as carfilzomib, and not with bortezomib. Citation Format: Kristine Misund, Katarzyna Anna Baranowska, Toril Holien, Kristian Starheim, Ida Johansson, Glenn Buene, Anders Waage, Geir Bjørkøy, Anders Sundan. Chloroquine potentiates carfilzomib but not bortezomib effects on myeloma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1768. doi:10.1158/1538-7445.AM2015-1768