Ixazomib Research Articles

Spirocyclic character of ixazomib citrate revealed by comprehensive XRD, NMR and DFT study

Ixazomib citrate is a very recently approved anti-cancer drug. Until now, to the best of our knowledge, no one has been able to solve any crystal structures of this compound. In this work, we present the crystal structures of two isostructural solvates of ixazomib citrate. In all currently available literature, the molecule is characterized as containing a single optically active carbon atom and a borate cycle formed when ixazomib is reacted with citric acid to form a stabilized ixazomib citrate that can be administered orally. However, the crystal structures revealed that none of the up-to-date presented structural formulas of ixazomib citrate are fully accurate. In addition to the citrate ring, another 5-membered ring is formed. These two rings are connected by the boron atom, making this compound a spirocyclic borate. By spirocyclization, the boron atom becomes tetrahedral and therefore optically active. In the crystal structures, ixazomib citrate was found to be in forms of two RR and RS stereoisomers. The results are supported by solid-state and solution NMR and DFT quantum mechanical calculations.

The evaluation of the anti-cancer activity of ixazomib on Caco2 colon solid tumor cells, comparison with bortezomib

Proteasome inhibition has recently emerged as a clinically effective anticancer therapeutic approach. The first proteasome inhibitor, bortezomib (Velcade, PS-341), and new proteasome inhibitors including ixazomib have become more important in the development of targeted cancer therapies. Under physiological conditions, MLN9708 (ixazomib citrate), the stable citrate ester drug substance, hydrolyzes rapidly to MLN2238 (ixazomib), the biologically active boronic acid. It is a second-generation proteasome inhibitor, similar to the well-known proteasome inhibitor bortezomib, which is currently being investigated in phase 3 trials as a treatment for multiple Myeloma. Despite the proven efficacy of these drugs in hematologic malignancies, clinical activity is limited to solid tumors such as colon adenocarcinoma. This study is the first to investigate and compare the antiproliferative and apoptotic effects of MLN2238 and bortezomib on human colon adenocarcinoma Caco2 cells. The antiproliferative effects of MLN2238 and bortezomib were determined using WST-1; apoptotic effects of this drug were determined by caspase-3 and a mitochondrial membrane potential (JC-1) activity assay. Expression levels associated with proteasome inhibition and apoptosis of NF-κB and c-myc mRNA were evaluated by RT-PCR. At 24 and 48 h, MLN2238 showed significant time- and concentration-dependent antiproliferative and apoptotic effects on Caco2 cells. Depending on increasing mitochondrial depolarization and caspase-3 activation, MLN2238 induced apoptosis at level similar to that of bortezomib. In addition, MLN2238 downregulated NF-κB and c-myc mRNA expression levels. For the first time, MLN2238 was shown to induce antiproliferative and apoptotic effects on human colon adenocarcinoma cells that are comparable with those of bortezomib; these in vitro data in Caco2 cells support the development of MLN2238 for colon cancer.

Treatment algorithms for multiple myeloma in Japan.

Recent progress in the development of novel therapeutic agents has remarkably improved the treatment outcome for multiple myeloma (MM). Proteasome inhibitors such as bortezomib, carfilzomib, and ixazomib; immunomodulatory drugs (IMiDs) such as thalidomide, lenalidomide, and pomalidomide; the histone deacetylase (HDAC) inhibitor panobinostat; and the monoclonal antibody, elotuzumab, have all been approved in Japan, although only bortezomib and lenalidomide have been approved for initial therapy. Accordingly, the Japanese Society of Hematology has released updated treatment guidelines for MM. Initial treatments with bortezomib-based 2-3 drug regimens and lenalidomide＋dexamethasone are recommended for patients eligible for autologous stem cell transplantation (ASCT), while bortezomib＋melphalan＋prednisolone and lenalidomide＋dexamethasone are recommended for patients who are not eligible for ASCT. These novel agents provide us with wider therapeutic options for relapsed or refractory patients. Consequently, treatment paradigms for MM continue to rapidly evolve, and it is important to select the optimal treatment strategy for each patient.

Proteasome inhibitors in first-line treatment of transplant-ineligible multiple myeloma patients.

Since the turn of the century, many agents against multiple myeloma (MM) have been introduced into daily clinical practice. The development of further agents is ongoing and some of these will reach the point of use in clinical practice in the near future. As various treatment options become available, the selection of an appropriate treatment strategy for an individual patient becomes more important. Treatment selection and decision making are based on the following two apparently opposite factors: 1) generalized findings and evidence from clinical trials, and 2) disease risks and background of individuals, which are diverse among patients. Proteasome inhibitors (PIs) are central players in MM treatment. In this review, we summarize evidence for the efficacy and safety of a first generation PI, bortezomib, and discuss its advantages in controlling disease risks and myeloma-defining events in patients with transplant-ineligible, untreated, newly diagnosed MM (NDMM). We also highlight on recent knowledge regarding the use of second generation PIs such as carfilzomib and ixazomib in the treatment of transplant-ineligible, untreated NDMM.

Counteracting PD-L1/PD-L2 Immune Suppression in Multiple Myeloma through Kinase Inhibition

Introduction: Multiple myeloma (MM) is clonal plasma cell malignancy that remains incurable. Checkpoint inhibitors represent a revolutionary form of cancer therapy that empowers the immune system to defeat cancer. Programmed death-1 (PD-1) is an inhibitory receptor expressed on immune cells, particularly cytotoxic T cells, that interacts with two ligands, PD-ligand 1 (PD-L1) and PD-L2 expressed on tumor cells. PD-L1 and PD-L2 engage PD-1 on the T cell surface to negatively modulate the magnitude of T-cell-mediated responses. Such negative feedback is critically important in maintaining homeostasis of the immune response to prevent autoimmunity during infection or inflammation in normal tissue. However, in cancers, it presents a major problem in blocking cellular antitumor responses. PD-L1/PD-L2 ligation with PD-1 provides a mechanism of immune escape for tumor cells by turning off the cytotoxic T cells. Currently available monoclonal antibodies (mAbs) that disrupt the PD-1/PD-L1 interaction have exhibited remarkable responses in selected patients and tumor types. However, mAbs demonstrate many drawbacks that include a lack of tumor cell specificity, low response rates in unselected patient populations and induction of de novoautoimmune disease that excludes many patients from therapy. We hypothesize that small molecule checkpoint inhibitors can provide greater specificity, shortened half-lives to diminish autoimmune or other adverse events, increased oral bioavailability, enhanced bio-efficacy, and higher stability at ambient temperature facilitating purification during production. Here, we identified novel, small molecule PI3K inhibitors that reduce PD-L1/PD-L2 levels on MM cells and enhance the antimyeloma activity of autologous T cells.Methods: To determine the effect of proteasome inhibitors on PD-L1/PD-L2 surface expression, RPMI8226 cells were incubated with bortezomib (BTZ), carfilzomib (CFZ) or ixazomib (IXZ) (1nM) for 36 h. Cells were then incubated in PBS/10% normal goat serum followed by antibodies (1/100) for 30 min at 4ºC and then treated with Alexa-647-conjugated anti-PD-L1 or FITC-conjugated anti-PD-L2. RPMI8226 cells were also incubated with the small molecule PI3K inhibitor DT97 (500nM) alone or combined with BTZ, CFZ, or IXZ. Cells were similarly stained using Alexa-647-conjugated anti-PD-L1 or FITC-conjugated anti-PD-L2 and a BD LSRFortessa™ cell analyzer was used for multicolor flow cytometry to acquire >10,000 events/sample. To determine the effect of DT97 on T cell-mediated cytolysis, bone marrow biopsy was performed on an MM patient and CD138+ cells were isolated by Miltenyi positive selection and CD4+ T cells by MojoSort™ CD4+ Human T Cell isolation. CD138+ cells were incubated with CD4+ T cells, proteasome inhibitors, or DT97 as indicated for 16 h at 37ºC. CD138+cells were then affinity-isolated, incubated with a FITC-conjugated anti-Annexin-V antibody and quantitated using a BD LSRFortessa™ cell analyzer.Results: Treatment of MM cells with BTZ, CFZ, or IXZ significantly increased the surface expression of PD-L1 and PD-L2 (Fig. 1). In contrast, treatment with the PI3K inhibitor DT97 suppressed the expression of PD-L1 and PD-L2 on MM cells (Fig. 2). Importantly, DT97 co-treatment with either BTZ, CFZ, or IXZ suppressed the induction of PD-L1 and PD-L2 seen after treatment with proteasome inhibitors alone. Treatment with the proteasome inhibitors alone or combined with T cells did not promote significant killing of MM cells (Fig. 3). However, DT97 treatment significantly enhanced autologous T cell-mediated MM death and the effect was further enhanced by addition of proteasome inhibitors.Conclusions: The results presented here establish the proof-of-principle that small molecule PI3K inhibitors reduce PD-L1/PD-L2 levels on tumor cells and enhance the anti-myeloma effect of autologous T cells. Small molecules checkpoint inhibitors represent a safe therapeutic alternative that can avoid the problems associated with antibodies, while retaining their functionality. Taken together, our studies indicate the promise of small molecules checkpoint inhibitors and support further translational and clinical development as a transformative form of cancer immunotherapy. DisclosuresNo relevant conflicts of interest to declare.

Importance of pharmacovigilance in the era of small molecules: Role of pharmacist consultation with ixazomib (IXA) in multiple myeloma (MM).

8058Background: IXA is an oral proteasome inhibitor recently approved for MM. Due to IXA’s Cytochrome P450 CYP3A4 and 1A2 metabolism, clinical trials of IXA excluded pts on 3A4 and 1A2 strong inhibitors and inducers. We examined the role of pharmacist review of potential drug-drug interactions (DDIs) in patients (pts) initiating IXA. Methods: Clinical trial pts receiving IXA between 1/2012 and 12/2015 for relapsed MM (NCT01415882; IXA, dexamethasone) and newly diagnosed MM (NCT01864018; IXA, dexamethasone, cyclophosphamide) were included. A pharmacist medication review for DDI was performed prior to IXA initiation. Baseline characteristics, concomitant medications, and toxicities were recorded. Results: Ninety-seven pts were included; 1 pt was excluded due to use of clarithromycin, a strong CYP3A4 inhibitor. Median age was 69 years (41-88) and 57% were male. The median number of concomitant medications was 9 (1-23). Four (4%) pts had potential CYP-mediated DDIs. One pt was on a strong CYP3A4 inducer (prim...

Oral Triplet Therapy for Relapsed or Refractory Myeloma

Triplet therapy with an immunomodulatory drug, a proteasome inhibitor, and dexamethasone has improved response and progression-free survival in patients with multiple myeloma. To test the activity of the oral proteasome inhibitor ixazomib in myeloma triplet therapy, investigators conducted an industry-sponsored, multicenter, randomized, double-blind, placebo-controlled phase III trial comparing lenalidomide plus dexamethasone with either ixazomib or placebo in 772 patients …

Current treatment of refractory and relapsed multiple myeloma.

In the past decade, previously approved novel agents, such as proteasome inhibitors (bortezomib) and immunomodulatory drugs ([IMiDs]; e.g., lenalidomide), have led to significant improvement in the treatment of multiple myeloma in Japan. However, almost all patients will ultimately relapse, even when they have achieved a deep and prolonged therapeutic response with initial treatment. Next-generation IMiDs (pomalidomide) and deacetylase inhibitors (panobinostat) were approved for use as salvage therapy for refractory and relapsed multiple myeloma [RRMM] within the last year. Long-term chemotherapy could result in the emergence of drug-resistant clones due to "intraclonal heterogeneity" and "clonal evolution by Darwinian selection." Though some recommendations on the management of RRMM have been detailed, no uniform treatment has yet been established for these patients. Relapse situations are heterogeneous. Therefore, relapse management requires an individual approach based on assessments of patient-, disease-, and treatment-related factors. The primary considerations when selecting an appropriate treatment are patient-related factors such as frailty, comorbidity, disability, quality of life, and the overall goals of care. We hope that these novel agents that appear promising in Japan, such as monoclonal antibodies (e.g., elotuzumab, daratumumab) and next-generation proteasome inhibitors (e.g., carfilzomib, ixazomib) will improve the outcomes of patients with this incurable disease in the near future.

Ixazomib Impairs Dendritic Cell Function and T Cell Proliferation and Affects the Development of GvHD in a Schedule-Dependent Fashion

Background: Targeting T cells alone has yielded limited success in the prevention of graft-versus-host disease (GvHD) following allogeneic blood and marrow transplantation (BMT). Dendritic cells (DCs) play a central role in alloreactivity and therefore represent a suitable target. Proteasome inhibitors (PI), with their ability to inhibit the function and maturation of DC, have prompted investigators to examine their potential role in the prevention of GvHD. The investigational PI, ixazomib (IXZ), dissociates rapidly from 20S and is therefore truly reversible. It is also orally bioavailable. Our aim in this study was to explore its effect on healthy volunteer peripheral blood dendritic and T cells and in a pre-clinical GvHD mouse model.Methods: To characterize the effects of IXZ on healthy volunteer peripheral blood DCs, DCs were isolated using EasySep Pan-DC Pre-Enrichment Cocktail with purity over 90% (STEMCELL Technologies). DCs were then treated with IXZ at different concentrations (10-40nM) for 4 hrs and then stimulated with lipopolysaccharide (LPS) for 16 hrs. After this treatment, DCs were surface stained with antibodies against maturation markers and analyzed by flow cytometry. DC survival was evaluated with 7AAD staining and FACS analysis. To assess the effect of IXZ on the production of pro-inflammatory cytokines, DCs were incubated with IXZ at increasing concentration before or after the addition of LPS. Total pro-inflammatory cytokines in the supernatant of tissue culture were measured using EMD Millipore cytokine arrays. Standard mixed lymphocyte reaction and T cell proliferation assays were used to evaluate T cell function. At a minimum, all experiments were done in triplicate. Unpaired t test was used for statistical analysis. A p-value < 0.05 was considered significant. The B6 → BALB/c pre-clinical GvHD model was adopted to evaluate the effect of IXZ on GvHD development. Mice were transplanted in 3 groups. Group 1 received a lethal dose of total body irradiation (TBI), donor bone marrow (BM) cells, and IXZ, group 2 received TBI, donor BM cells donor splenocytes, and a vehicle, and group 3 received TBI, donor BM cells, donor splenocytes, and IXZ. The dose of BM cells and splenocytes was 5 X 106 each. IXZ was given at 1.5 mg/kg subcutaneously. Two dosing schedules were tested in 2 separate experiments: day-1 and +2 or day +1 and +4.Results: IXZ inhibited the expression of 6 DC maturation markers including CD40, CD54, CD80, CD83, CD86 and CD197 (CCR-7). The inhibition started at a concentration of 10nM and was dose-related. IXZ also decreased the percentage of total DCs simultaneously expressing multiple markers. DCs viability remained unchanged in comparison to control at a concentration of 10nM and dropped to 68% and 43%, on average with concentrations of 20nM and 40nM, respectively. IXZ significantly decreased DC production of IL-6, IL-12, and IL-23 starting at the concentration of 20nM. IL-1β was decreased at the concentration of 40 nM. Importantly, there was no significant change in the cytokine production by DCs when IXZ was added 4 hrs after LPS except for IL-1β which increased at 30nM. Starting at the concentration of 10nM, IXZ dose-dependently inhibited T cell proliferation. At 40nM IXZ abolished T cells. In our in vivo study IXZ improved GvHD scores on days +7 and +11 in group 3 in comparison to group 2 when it was given on days -1 and +2. Conversely, when IXZ was given on day +1 and +4, group 3 mice had higher scores of GvHD and worse survival outcomes when compared to group 2. There was no noticeable drug toxicity in group 1 mice.Conclusion: In summary: 1) IXZ inhibits DC maturation with relative preservation of cell viability and inhibits pro-inflammatory cytokine production in DCs when added before LPS stimulation; 2) IXZ inhibits T-cell proliferation; 3) IXZ affects GvHD development in a schedule-dependent fashion with early administration improving and late administration worsening GvHD. Additional analysis of tissue and serum samples is in progress. These results provide background for careful design of clinical trials using IXZ for the prevention of GvHD. DisclosuresAl-Homsi:Millennium Pharmaceuticals: Research Funding.

Abstract B147: A phase 1 drug-drug interaction study between ixazomib, an oral proteasome inhibitor, and rifampin in patients (pts) with advanced solid tumors

Abstract Background Ixazomib is an oral proteasome inhibitor under phase 3 investigation in pts with multiple myeloma and AL amyloidosis. Ixazomib citrate, a prodrug, rapidly hydrolyzes to the active moiety, ixazomib, in plasma. Metabolism appears to be the major route of elimination for ixazomib. This phase 1, open-label, multicenter, parallel-group study (NCT01454076) investigated the effect of rifampin, an established strong CYP3A inducer, on the pharmacokinetics (PK) of ixazomib. Methods Adult pts with advanced solid tumors (Eastern Cooperative Oncology Group Performance Status 0 or 1), for which no effective standard treatment was available, were enrolled. Pts in the ixazomib + rifampin arm received a single 4 mg dose of ixazomib on day 8, plus rifampin 600 mg PO on days 1-14 of a 21-day PK cycle. On day 8, ixazomib and rifampin were administered concomitantly and PK samples were collected over 168 hours post-dose. Pts in the reference arm received a single 4 mg dose of ixazomib on day 1 with PK samples collected over 168 hours post-dose. After completion of the 21-day PK cycle, pts could continue in the study and receive ixazomib on days 1, 8, and 15 of 28-day cycles. Plasma PK parameters were estimated by non-compartmental methods. Geometric mean ratios and 90% confidence intervals (CIs) of PK parameters in the ixazomib + rifampin versus ixazomib alone arms were calculated using an ANOVA model. Treatment-emergent adverse events (TEAEs) were assessed using NCI CTCAE version 4.03. Results Eighteen and 20 pts were enrolled to the ixazomib + rifampin and ixazomib alone arms, respectively. To assess the impact of rifampin on ixazomib PK, data from 16 PK-evaluable pts who received ixazomib + rifampin were compared with data from 14 PK-evaluable pts who received ixazomib alone. Demographics and PK parameters are shown in the Table. At data cut-off (4 August 2014), 15 pts (83%) in the ixazomib + rifampin arm had ≥1 TEAE related to study medication. The most common (≥20%) drug-related TEAEs, regardless of grade, were nausea (39%) and fatigue (28%). Three pts (17%) in the ixazomib + rifampin arm experienced ≥1 grade 3 TEAE. Conclusions The strong CYP3A inducer rifampin produced a 74% decrease in ixazomib total systemic exposure. Systemic treatment with strong CYP3A inducers should be avoided in pts receiving ixazomib. ParameterIxazomib + rifampin (test)Ixazomib alone (reference)LS geometric mean ratio (90% CI) (test / reference)N (PK evaluable)18 (16)20 (14)-Age, yearsa62 (39-81)61 (29-76)-Male,%5645-Weight, kga80.2 (58.9-109.3)69.8 (46.9-93.5)-Tmax, hoursb1.45 (0.5-4.12)1.49 (0.5-7.5)-Cmax, ng/mLc25.7 (50)55.8 (57)0.46 (0.29, 0.73)AUC0-last, ng•hr/mLc232 (50)907 (44)0.26 (0.18, 0.37)aMean (range); bmedian (range); cgeometric mean (% coefficient of variation). AUC, area under the plasma concentration-time curve from time 0 to the time of the last quantifiable concentration; CI, confidence interval; Cmax, maximum plasma concentration; LS, least square; PK, pharmacokinetic; Tmax, time to first maximum plasma concentration. Citation Format: Neeraj Gupta, Michael J. Hanley, Karthik Venkatakrishnan, Alberto Bessudo, Sunil Sharma, Bert O'Neil, Bingxia Wang, Ai-Min Hui, John Nemunaitis. A phase 1 drug-drug interaction study between ixazomib, an oral proteasome inhibitor, and rifampin in patients (pts) with advanced solid tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B147.

The investigational proteasome inhibitor ixazomib for the treatment of multiple myeloma.

Ixazomib is an investigational, reversible 20S proteasome inhibitor. It is the first oral proteasome inhibitor under clinical investigation in multiple myeloma (MM). Under physiological conditions, the stable citrate ester drug substance, ixazomib citrate (MLN9708), rapidly hydrolyzes to the biologically active boronic acid, ixazomib (MLN2238). Preclinical studies have demonstrated antitumor activity in MM cell lines and xenograft models. In Phase I/II clinical studies ixazomib has had generally manageable toxicities, with limited peripheral neuropathy observed to date. Preliminary data from these studies indicate ixazomib is active as a single agent in relapsed/refractory MM and as part of combination regimens in newly diagnosed patients. Phase III studies in combination with lenalidomide-dexamethasone are ongoing.

An evidence-based review of ixazomib citrate and its potential in the treatment of newly diagnosed multiple myeloma

Proteasome inhibition represents one of the more important therapeutic targets in the treatment of multiple myeloma (MM), since by suppressing nuclear factor-κB activity, which promotes myelomagenesis, it makes plasma cells susceptible to proapoptotic signals. Bortezomib, the first proteasome inhibitor approved for MM therapy, has been shown to increase response rate and improve outcome in patients with relapsed/refractory disease and in the frontline setting, particularly when combined with immunomodulatory drugs and alkylating agents. Among second-generation proteasome inhibitors, ixazomib (MLN9708) is the first oral compound to be evaluated for the treatment of MM. Ixazomib has shown improved pharmacokinetic and pharmacodynamic parameters compared with bortezomib, in addition to similar efficacy in the control of myeloma growth and prevention of bone loss. Ixazomib was found to overcome bortezomib resistance and to trigger synergistic antimyeloma activity with dexamethasone, lenalidomide, and histone deacetylase inhibitors. Phase I/II studies using ixazomib weekly or twice weekly in relapsed/refractory MM patients suggested antitumor activity of the single agent, but more promising results have been obtained with the combination of ixazomib, lenalidomide, and dexamethasone in newly diagnosed MM. Ixazomib has also been used in systemic amyloidosis as a single agent, showing important activity in this difficult-to-treat plasma-cell dyscrasia. More frequent side effects observed during administration of ixazomib were thrombocytopenia, nausea, vomiting, diarrhea, fatigue, and rash, whereas severe peripheral neuropathy was rare. Here, we review the chemical characteristics of ixazomib, as well as its mechanism of action and results from preclinical and clinical trials.

Ixazomib Citrate (MLN9708) Inhibits Kidney Allograft Infiltrating Macrophages in a CNI Minimizing Strategy.

Macrophages (MΦ) play an important role in acute rejection after kidney transplantation (Tx). Calcineurin inhibitor minimizing strategies may be associated with improved outcome after Tx. We sought to determine the role of a new protease inhibitor (PI, Ixazomib citrate, MLN9708) on MΦand MΦ associated genes in a CNI minimization strategy. Lewis (RT1l) recipients were transplanted with a full MHC mismatch Brown Norway (RT1n) rat kidney. Recipients were randomized into four groups: Control Brown Norway (C), Transplant with no treatment (Tx), transplant with full dose CsA (Tx, 10 mg/kg/d) or transplant with MLN9708 + half dose CsA (TxMLNCsA, 5 mg/kg/d). One week post-transplant, RNA was isolated from kidney for gene microarray analysis on Affymetrix Rat ST 2.0 chips. Differentially expressed gene (DEG) and pathway analyses were performed using iReport. Cellular analyses were performed using immunohistochemistry. Tx alone animals had a strong influx of MΦ into the kidney. Treatment with full dose CsA or low dose CsA + MLN9708 greatly reduced MΦ infiltration. The molecular signature associated with MΦ activation pathways was downregulated in animals treated with MLN9708 + ½ CsA, similar to full dose CsA.Table: No Caption available.These genes included IL-10, heme oxygenase 1 and biomarkers of M1 (iNOS) and M2 (arg1, CD163) MΦ phenotypes. These preclinical studies suggest that CNI minimization strategies with novel proteasome inhibitors may be effective in preventing macrophage-induced allograft injury. Further studies are needed to better characterize the significance and regulation of macrophage associated genes by proteasome inhibitors.

Integrated nonclinical and clinical risk assessment to obviate the need for a dedicated QTc study of ixazomib citrate (MLN9708) in cancer patients (pts).

2553 Background: Ixazomib citrate is an investigational oral proteasome inhibitor in phase 3 (P3) clinical development. It is expected to have a favorable cardiac safety profile based on weak in vi...

Preclinical Activity of the Oral Proteasome Inhibitor MLN9708 in Myeloma Bone Disease

MLN9708 (ixazomib citrate), which hydrolyzes to pharmacologically active MLN2238 (ixazomib), is a next-generation proteasome inhibitor with demonstrated preclinical and clinical antimyeloma activity, but yet with an unknown effect on myeloma bone disease. Here, we investigated its bone anabolic and antiresorptive effects in the myeloma setting and in comparison with bortezomib in preclinical models. The in vitro effect of MLN2238 was tested on osteoclasts and osteoclast precursors from healthy donors and patients with myeloma, and on osteoprogenitors derived from bone marrow mesenchymal stem cells also from both origins. We used an in vivo model of bone marrow-disseminated human myeloma to evaluate MLN2238 antimyeloma and bone activities. Clinically achievable concentrations of MLN2238 markedly inhibited in vitro osteoclastogenesis and osteoclast resorption; these effects involved blockade of RANKL (receptor activator of NF-κB ligand)-induced NF-κB activation, F-actin ring disruption, and diminished expression of αVβ3 integrin. A similar range of MLN2238 concentrations promoted in vitro osteoblastogenesis and osteoblast activity (even in osteoprogenitors from patients with myeloma), partly mediated by activation of TCF/β-catenin signaling and upregulation of the IRE1 component of the unfolded protein response. In a mouse model of bone marrow-disseminated human multiple myeloma, orally administered MLN2238 was equally effective as bortezomib to control tumor burden and also provided a marked benefit in associated bone disease (sustained by both bone anabolic and anticatabolic activities). Given favorable data on pharmacologic properties and emerging clinical safety profile of MLN9708, it is conceivable that this proteasome inhibitor may achieve bone beneficial effects in addition to its antimyeloma activity in patients with myeloma.

Highlights of This Issue

MLN9708 (ixazomib citrate) is an oral next-generation proteasome inhibitor (PI) currently in phase III trials in multiple myeloma patients, but with a yet-unknown effect on myeloma-associated bone disease. Garcia-Gomez and colleagues show that clinically relevant concentrations of ixazomib promote osteoblast differentiation and function and inhibit osteoclast formation and resorption in vitro. Also, in a disseminated myeloma mouse model, ixazomib controls myeloma growth and prevents bone loss, as assessed by bone-forming and anticatabolic activities. Thus, it is conceivable that MLN9708, similar to the first-in-class PI bortezomib, will achieve beneficial bone effects, in addition to antimyeloma activity in myeloma patients.Hedgehog-GLI signaling is upregulated in a proportion of lung squamous cell carcinoma (LSCC), but no studies to date evaluate its role in LSCC. Here, Huang and colleagues identified overexpression of HEDGEHOG-GLI components in the classical subtype of LSCC via genomic analysis. In representative cell lines, they demonstrated that GLI2 was important for LSCC cell survival, whereas SMOOTHENED plays a minor role. Importantly, they reported that the GLI inhibitor GANT61 successfully suppressed tumor growth both in vitro and in vivo and thus shows promise as a novel targeted therapy for a subset of LSCC patients.The combination therapy of sorafenib and TACE is considered an alternative for patients with unresectable HCC. The assessment of tumor response to this therapy is particularly crucial in determining treatment success. To define the earliest time at which the response to combination therapy could be accurately assessed, Liu and colleagues demonstrated that the available criteria EASL and mRECIST responses assessed after 3 to 4 months of therapy are more reliable for detecting the early changes in the tumor and can be considered valuable early indicators for making subsequent therapeutic decisions and predicting long-term survival.Analysis of cell-free plasma DNA represents an attractive strategy for noninvasive genotyping of advanced cancer patients. Oxnard and colleagues designed quantitative plasma-based genotyping assays for EGFR, KRAS, and BRAF mutations using droplet digital PCR and demonstrated a high positive predictive value in patients with advanced lung cancer and melanoma. Serial analysis of plasma from lung cancer patients on a prospective trial of first-line erlotinib demonstrated high pretreatment EGFR levels, complete plasma response, and detection of a T790M resistance mutation weeks prior to clinical progression, suggesting potential as a noninvasive clinical tool for characterizing treatment response and resistance.

Twice-Weekly Oral MLN9708 (Ixazomib Citrate), An Investigational Proteasome Inhibitor, In Combination With Lenalidomide (Len) and Dexamethasone (Dex) In Patients (Pts) With Newly Diagnosed Multiple Myeloma (MM): Final Phase 1 Results and Phase 2 Data

Twice-Weekly Oral MLN9708 (Ixazomib Citrate), An Investigational Proteasome Inhibitor, In Combination With Lenalidomide (Len) and Dexamethasone (Dex) In Patients (Pts) With Newly Diagnosed Multiple Myeloma (MM): Final Phase 1 Results and Phase 2 Data

Evaluation Of Potency and The Associated Biology Of The Investigational Proteasome Inhibitor, Ixazomib: Redox, Autophagic, and MAPK-Dependent Cell Death In T-Cell Lymphoma (TCL) and Hodgkin Lymphoma (HL) Cell Lines and Human Lymphoma Xenograft Models

Background We investigated the antitumor activity and biologic mechanisms of action of the investigational proteasome inhibitor, ixazomib citrate (MLN2238), in TCL and HL cells with in vitro and in vivo tumor models. Methods TCL cell lines (Jurkat, Hut78 and HH) and HL cell lines (L428, L540, L1236) were treated with ixazomib for 24-48 hours. We analyzed apoptosis and oxidative stress by flow cytometry (FC), and expression of p21, MYC, and MAPK were analyzed with Western blot. For analysis of autophagy, activation of lysosome was examined by fluorescence microscopy using monodansylcadaverine (MDC), which was confirmed by Western blot analysis for Cathepsin D activation and electron microscopy. Additionally, we interrogated signaling pathways with stably transfected shRNA knockouts (KO) +/- ixazomib. In vivo tumor growth inhibition and survival of tumor bearing SCID mice were determined using xenografts derived from Jurkat (TCL) and L540 (HL) cell lines. Results Treatment with 50-100 nanomolar (nM) of ixazomib resulted in time- and dose-dependent cytotoxicity in all cell lines. The IC50 values were 38nM, 52nM, and 41nM for Jurkat, Hut78, and HH, respectively, and 39nM, 60nM, and 117nM for L540, L1236, and L428, respectively. Further, ixazomib resulted in dose-dependent increases in apoptosis by Annexin-V/PI (P&lt;0.001), cleaved PARP, and activation of caspases 3, 8, and 9 in all cell lines. Ixazomib also resulted in increased oxidative stress and decreased intra-cellular glutathione (GSH) and it caused increased p21 expression and degradation of MYC protein. These effects appeared to be redox-dependent as treatment with N-acetyl cysteine (NAC) abrogated apoptosis, oxidative stress, and MYC degradation. Moreover, treatment with ixazomib resulted in cytoplasmic lysosomal abundance with localization in the perinuclear and nuclear region, as observed by MDC staining and electron microscopy in Jurkat and L428. Western blot analysis showed activation of Cathepsin D and presence of Cathepsin D in the nuclear fraction with ixazomib treatment. Furthermore, the autophagic inhibitor, spautin, abrogated cell death induced by ixazomib suggesting a critical role for lysosome directed cytotoxicity with ixazomib-induced cell death. In addition, ixazomib induced several changes in MAPK signaling. In TCL, it increased pERK and p-p38, while pJNK was decreased. Using shRNAs (with ixazomib), ERK and p38 KO had minimal effect in the TCL lines, however JNK KO resulted in increased cell death in Jurkat and Hut78 cells. In HL, ERK and JNK shRNA had minimal effect, while p38 KO increased the cytotoxic effect of ixazomib in all HL cell lines. Finally, in vivo experiments with Jurkat and L540 xenograft-bearing SCID mice showed that ixazomib treatment resulted in significant inhibition of tumor growth (P&lt;0.001) as well as significantly improved survival (P&lt;0.001) versus controls (Figure 1). Conclusions Altogether, ixazomib induced potent cell death at nanomolar and clinically achievable concentrations in TCL and HL cell lines and in vivo human xenograft models. In all cell lines, ixazomib down-regulated MYC and cell death was redox-dependent. Further, autophagy appeared to strongly mediate apoptosis through lysosome directed cytotoxic response. Additionally, in TCL, the cytotoxic effect of ixazomib was mediated through JNK, while p38 was the dominant pathway in HL-related cell death. Continued examination of this novel agent in lymphoma is warranted and rational combinations with MAPK inhibitors should be explored. Disclosures: Evens: Millennium: Consultancy, Honoraria.

Preclinical Combination Of The Oral Investigational Agents ACY-1215, a Selective HDAC6 Inhibitor, and Ixazomib, a Proteasome Inhibitor, Demonstrates Combination Benefit In Multiple Myeloma Cell Lines and Xenograft Models

Introduction The combination of HDAC inhibitors and proteasome inhibitors has demonstrated preclinical benefit in several settings, including multiple myeloma and lymphoma, and is being explored in clinical trials testing various HDAC inhibitors in combination with proteasome inhibitors. ACY-1215 is an investigational, orally available HDAC6-selective inhibitor that has demonstrated preclinical combination benefit with bortezomib in vitro and in vivo (Santos et al, Blood 2012; 119: 2579). These preclinical studies also support the hypothesis that the improved selectivity of ACY-1215 for HDAC6 over class I HDACs (HDAC1,2,3) may provide an improved tolerability profile compared to pan-HDAC inhibitors, while still providing the anti-myeloma effect of other HDACi/proteasome inhibitor combinations. ACY-1215 is currently in a Phase I/II trial in multiple myeloma with bortezomib (VELCADE) and dexamethasone to test this hypothesis (NCT01323751). Ixazomib citrate (MLN9708) is an investigational oral proteasome inhibitor in Phase III clinical trials in multiple myeloma (NCT01850524, NCT01564537). To examine the potential efficacy of the all-oral combination of ixazomib citrate and ACY-1215, we evaluated the combination of these agents in cell lines and xenograft models of multiple myeloma. Results In vitro viability experiments in 2 multiple myeloma cell lines (RPMI-8226 and MM.1S) using a dose matrix format demonstrated a combination benefit of ACY-1215 and ixazomib over a range of concentrations, very similar to the previously reported benefit of ACY-1215 plus bortezomib. Likewise, the combination benefit of the selective HDAC6 inhibitor ACY-1215 with ixazomib was similar to the combination effect observed with the pan-HDAC inhibitor SAHA (vorinostat). Together, these in vitro studies support the hypothesis that the combination of ACY-1215 and ixazomib provides similar levels of benefit as do combinations including other HDACi/proteasome inhibitors. Furthermore, experiments in MM.1S xenograft-bearing mice demonstrated an in vivo combination benefit of ACY-1215 and ixazomib. An all-oral regimen was well tolerated when ACY-1215 was dosed at 100 mg/kg PO twice daily for 5 days per week in combination with ixazomib dosed at 5 mg/kg PO twice weekly, and the combination regimen demonstrated additive antitumor activity (Figure 1). The in vivo combination benefit of ACY-1215 and ixazomib was further demonstrated in MM.1S xenograft-bearing mice using alternate routes of administration (IV dosing of ixazomib and IP dosing of ACY-1215). The combination of ACY-1215 dosed at 30 mg/kg IP once daily for 5 days per week with ixazomib dosed IV at 1.5 mg/kg twice-weekly was also well tolerated and had striking antitumor activity. This combination regimen in fact caused regression of the MM.1S xenograft tumors below the starting volumes, and this level of activity was maintained throughout the entire 17 day dosing period (Figure 2). In an accompanying pharmacodynamic (PD) study of the PO and IP doses of ACY-1215, we confirmed selective HDAC6 inhibition in MM.1S xenograft tumors as evidenced by elevated acetylation levels of the HDAC6 substrate tubulin, with little if any change in the levels of acetylated histone H3, a class I HDAC substrate. In vivo experiments in a second xenograft model, RPMI-8226, also demonstrated a combination benefit of ACY-1215 (30 mg/kg IP for 5 days per week) with ixazomib (0.75 mg/kg IV twice-weekly). Conclusion The combination benefit of ACY-1215 and ixazomib observed here in preclinical experiments utilizing in vitro and in vivo models of multiple myeloma provides rationale for clinical evaluation of this first all-oral combination of a proteasome inhibitor with an HDAC inhibitor. Disclosures: Berger: Takeda Pharmaceutical Company Ltd: Employment. Bannerman:Takeda Pharmaceutical Company Ltd: Employment. Quayle:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Yu:Takeda Pharmaceutical Company Ltd: Employment. Garcia:Takeda Pharmaceutical Company Ltd: Employment. Ciavarri:Takeda Pharmaceutical Company Ltd: Employment. Tamang:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Yang:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Jones:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership.

18F-FDG-PET/CT imaging in an IL-6- and MYC-driven mouse model of human multiple myeloma affords objective evaluation of plasma cell tumor progression and therapeutic response to the proteasome inhibitor ixazomib

18F-fluorodeoxyglucose positron emission tomography (FDG-PET) and computed tomography (CT) are useful imaging modalities for evaluating tumor progression and treatment responses in genetically engineered mouse models of solid human cancers, but the potential of integrated FDG-PET/CT for assessing tumor development and new interventions in transgenic mouse models of human blood cancers such as multiple myeloma (MM) has not been demonstrated. Here we use BALB/c mice that contain the newly developed iMycΔEμ gene insertion and the widely expressed H2-Ld-IL6 transgene to demonstrate that FDG-PET/CT affords an excellent research tool for assessing interleukin-6- and MYC-driven plasma cell tumor (PCT) development in a serial, reproducible and stage- and lesion-specific manner. We also show that FDG-PET/CT permits determination of objective drug responses in PCT-bearing mice treated with the investigational proteasome inhibitor ixazomib (MLN2238), the biologically active form of ixazomib citrate (MLN9708), that is currently in phase 3 clinical trials in MM. Overall survival of 5 of 6 ixazomib-treated mice doubled compared with mice left untreated. One outlier mouse presented with primary refractory disease. Our findings demonstrate the utility of FDG-PET/CT for preclinical MM research and suggest that this method will play an important role in the design and testing of new approaches to treat myeloma.