Proteasome Inhibitor Ixazomib Research Articles

Selective Toxicity of Investigational Ixazomib for Human Leukemia Cells Expressing Mutant Cytoplasmic NPM1: Role of Reactive Oxygen Species.

This study was performed to determine whether the investigational proteasome inhibitor ixazomib demonstrated selective antineoplastic activity against acute myelogenous leukemia cells expressing a mutated nucleophosmin-1 gene and to gain a better understanding of its mechanisms of action. The cytotoxic effects of ixazomib treatment were analyzed in human acute myelogenous leukemia (AML) cell lines and primary AML samples expressing wild-type or mutated NPM1 (NPMc(+)). The potential roles of oxidative stress in mediating cytotoxic activity were determined using flow cytometry, enzyme-based assays, and Western blots. Apoptosis induced by ixazomib was abrogated by knockdown of NPM1/NPMc(+)expression using an inducible shRNA construct and enhanced by NPMc(+)overexpression. Cytotoxicity was associated with superoxide generation and was reduced by the addition of the antioxidant N-acetylcysteine. AML cells expressing NPMc(+)had significantly reduced levels of intracellular glutathione and NADPH associated with reduced antioxidant responses to drug treatment. Treatment of 3 patients with relapsed NPMc(+)AML resulted in an antileukemic effect in 1 patient as demonstrated by a marked reduction of leukemic blasts in the peripheral blood. Efficacy was associated with superoxide generation, reduced glutathione levels, and reduced mRNA and protein expression of antioxidant effectors in responding cells. In this study, a direct association was observed between NPMc(+)expression in AML, reduced antioxidant responses, and enhanced sensitivity to an oral proteasome inhibitor that induces oxidative stress. These data suggest that intracellular determinants of antioxidant responses may be good predictors of therapeutic response to ixazomib.

KRAS Genotype Correlates with Proteasome Inhibitor Ixazomib Activity in Preclinical In Vivo Models of Colon and Non-Small Cell Lung Cancer: Potential Role of Tumor Metabolism

In non-clinical studies, the proteasome inhibitor ixazomib inhibits cell growth in a broad panel of solid tumor cell lines in vitro. In contrast, antitumor activity in xenograft tumors is model-dependent, with some solid tumors showing no response to ixazomib. In this study we examined factors responsible for ixazomib sensitivity or resistance using mouse xenograft models. A survey of 14 non-small cell lung cancer (NSCLC) and 6 colon xenografts showed a striking relationship between ixazomib activity and KRAS genotype; tumors with wild-type (WT) KRAS were more sensitive to ixazomib than tumors harboring KRAS activating mutations. To confirm the association between KRAS genotype and ixazomib sensitivity, we used SW48 isogenic colon cancer cell lines. Either KRAS-G13D or KRAS-G12V mutations were introduced into KRAS-WT SW48 cells to generate cells that stably express activated KRAS. SW48 KRAS WT tumors, but neither SW48-KRAS-G13D tumors nor SW48-KRAS-G12V tumors, were sensitive to ixazomib in vivo. Since activated KRAS is known to be associated with metabolic reprogramming, we compared metabolite profiling of SW48-WT and SW48-KRAS-G13D tumors treated with or without ixazomib. Prior to treatment there were significant metabolic differences between SW48 WT and SW48-KRAS-G13D tumors, reflecting higher oxidative stress and glucose utilization in the KRAS-G13D tumors. Ixazomib treatment resulted in significant metabolic regulation, and some of these changes were specific to KRAS WT tumors. Depletion of free amino acid pools and activation of GCN2-eIF2α-pathways were observed both in tumor types. However, changes in lipid beta oxidation were observed in only the KRAS WT tumors. The non-clinical data presented here show a correlation between KRAS genotype and ixazomib sensitivity in NSCLC and colon xenografts and provide new evidence of regulation of key metabolic pathways by proteasome inhibition.

A Phase 1 Study to Assess Pharmacokinetics (PK) and Safety of Ixazomib, an Oral Proteasome Inhibitor, in Patients with Moderate or Severe Hepatic Impairment

BackgroundThe investigational oral proteasome inhibitor ixazomib is under phase 3 clinical investigation in patients with multiple myeloma and amyloidosis. Metabolism is the major mechanism of ixazomib clearance; accordingly, hepatic impairment may increase ixazomib exposures. PK and safety data suggested no clinically relevant PK alterations in patients with mild hepatic impairment (Gupta et al BJCP 2015). This study (NCT01912222) was performed to characterize the PK of ixazomib in patients with moderate or severe hepatic impairment, as defined by the NCI Organ Dysfunction Working Group, to develop dosing recommendations for these specific patient populations.MethodsEligible adults had advanced malignancies for which no further effective therapy was available. Twelve PK-evaluable patients were planned to be enrolled to each of the normal (N), moderate (M) or severe (S) hepatic impairment groups. Patients received a single dose of ixazomib on day 1; those in the N, M, and S groups received ixazomib 4, 2.3, and 1.5 mg, respectively. Blood samples were collected at multiple time points for 15 days after dosing to characterize single dose PK (18 samples in total, 0.5-8 hr on day 1, days 2-8, days 11, 12, and 15). After completion of PK sampling, patients could continue on the study and receive ixazomib on days 1, 8, and 15 of 28-day cycles. Geometric mean ratios and 90% CIs of unbound dose-normalized (DN) PK parameters in the hepatic impairment vs normal groups were calculated using an ANOVA model. Treatment-emergent adverse events (TEAEs) were assessed using NCI CTCAE version 4.03.ResultsForty-eight patients were enrolled (13, 15, and 20 patients to the N, M, and S groups, respectively); 32 were Caucasian, 10 African American, 2 Asian, and 4 other. Mean age was 56 years (range 24-83), mean weight 76 kg (range 43-127), and mean body surface area 1.9 m2 (range 1.4-2.5); 28 (58%) were male. The most common cancers were hepatocellular carcinoma (21%) and colorectal carcinoma, with or without liver metastases (10%). All patients in the S group had liver dysfunction due to primary or metastatic tumors. Forty-three patients had reportable PK parameters (Cmax or AUC) and were PK-evaluable (12 N, 13 M, 18 S). PK parameters are reported in the Table. Ixazomib was rapidly absorbed in all 3 hepatic function groups examined, with a median Tmaxof 0.95-1.5 hours. Ixazomib was highly bound to plasma proteins with a similar mean fraction bound (~99%) in all 3 groups.Only 1 (2%) patient continued on study for more than 3 cycles (endometrial carcinoma, 10 cycles). Discontinuations were due to progression (75% of discontinuations), TEAEs or death related to progression. The most common TEAEs were nausea (38%), fatigue (31%), peripheral edema (31%), vomiting (27%), dyspnea (23%), decreased appetite (21%), and hyperbilirubinemia (21%). Most patients (77%) had a grade ≥3 TEAE, including 15% with a grade ≥3 study drug-related TEAE (dehydration [6%], fatigue [4%], anemia [2%], and fall [2%]); no patient had a study drug-related grade 4 TEAE. There were 14 on-study deaths, all of which were considered related to disease progression.ConclusionsUnbound systemic exposures of ixazomib were 27% higher in patients with moderate or severe hepatic impairment versus those in patients with normal hepatic function. A reduced starting ixazomib dose of 3 mg is recommended for patients with moderate or severe hepatic impairment.TableHepatic function groupPK parameters Geometric mean (%CV)Hepatic impaired vs Normal Least-squares geometric mean ratio (90% CI)Unbound DN Cmax(ng/mL/mg)Unbound DN AUC (ng.hr/mL/mg)Unbound DN CmaxUnbound DN AUCNormal0.127 (47)2.41 (50)N/AN/AModerate0.162 (80)3.19 (61)1.27 (0.74-2.18)1.32 (0.70-2.50)Severe0.154 (84)2.96 (63)1.21 (0.74-2.01)1.23 (0.66-2.29)Moderate/Severe (combined)0.158 (81)3.07 (61)1.24 (0.79-1.95)1.27 (0.75-2.16)Cmax, maximum plasma concentration; CV, coefficient of variation; AUC, area under the plasma concentration-time curve DisclosuresGupta:Millennium Pharmaceuticals, Inc., Cambridge, MA, USA, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Off Label Use: Investigational proteasome inhibitor ixazomib. Hanley:Millennium Pharmaceuticals, Inc., Cambridge, MA, USA, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Venkatakrishnan:Millennium Pharmaceuticals, Inc., Cambridge, MA, USA, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Perez:Dompe: Research Funding; Eli Lilly: Research Funding; Incyte: Research Funding; Medimmune: Research Funding; Novartis: Research Funding; Pfizer: Research Funding; Millennium Pharmaceuticals Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Research Funding; Immunogen: Research Funding; Bristol Meyers Squibb: Research Funding; Agensys: Research Funding; PRA: Consultancy; Tetralogics: Research Funding. Norris:Nektar Pharmaceuticals: Consultancy. Yang:Millennium Pharmaceuticals, Inc., Cambridge, MA, USA, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Falchook:Millennium Pharmaceuticals, Inc., Cambridge, MA, USA, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Research Funding. Labotka:Millennium Pharmaceuticals, Inc., Cambridge, MA, USA, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment.

Phase 1/1b Pharmacokinetic (PK) and Safety Study of the Investigational Oral Proteasome Inhibitor (PI) Ixazomib in Relapsed/Refractory Multiple Myeloma (RRMM) Patients (Pts) with Severe Renal Impairment or End-Stage Renal Disease (ESRD) Requiring Hemodialysis

Phase 1/1b Pharmacokinetic (PK) and Safety Study of the Investigational Oral Proteasome Inhibitor (PI) Ixazomib in Relapsed/Refractory Multiple Myeloma (RRMM) Patients (Pts) with Severe Renal Impairment or End-Stage Renal Disease (ESRD) Requiring Hemodialysis

Randomized Phase 2 Study of the All-Oral Combination of Investigational Proteasome Inhibitor (PI) Ixazomib Plus Cyclophosphamide and Low-Dose Dexamethasone (ICd) in Patients (Pts) with Newly Diagnosed Multiple Myeloma (NDMM) Who Are Transplant-Ineligible (NCT02046070)

Randomized Phase 2 Study of the All-Oral Combination of Investigational Proteasome Inhibitor (PI) Ixazomib Plus Cyclophosphamide and Low-Dose Dexamethasone (ICd) in Patients (Pts) with Newly Diagnosed Multiple Myeloma (NDMM) Who Are Transplant-Ineligible (NCT02046070)

Ixazomib, an Investigational Oral Proteasome Inhibitor (PI), in Combination with Lenalidomide and Dexamethasone (IRd), Significantly Extends Progression-Free Survival (PFS) for Patients (Pts) with Relapsed and/or Refractory Multiple Myeloma (RRMM): The Phase 3 Tourmaline-MM1 Study (NCT01564537)

Ixazomib, an Investigational Oral Proteasome Inhibitor (PI), in Combination with Lenalidomide and Dexamethasone (IRd), Significantly Extends Progression-Free Survival (PFS) for Patients (Pts) with Relapsed and/or Refractory Multiple Myeloma (RRMM): The Phase 3 Tourmaline-MM1 Study (NCT01564537)

Proteasome Inhibitor-Adapted Myeloma Cells That Lack Proteasome Gene Mutations Resist Highly Efficient Proteasome Inhibition and Show Proteomic Alterations That Suggest Complex Metabolic Changes

Adaptive resistance of myeloma cells to proteasome inhibition is poorly understood. It is suggested to base on point mutations in PSMB5 and/or downmodulation of the activation state of the unfolded protein response (UPR) via reduced activity of its major regulatory axis IRE-1/XBP-1. We have generated subclones of the AMO-1 myeloma cell line resistant to bortezomib > 1000 nM (AMO-BTZ), or carfilzomib > 1000 nM (AMO-CFZ), that do or do not carry the PSMB5 A310G mutation in the β5 substrate pocket. We combine this model with a global quantitative proteomics approach, the analysis of the activation status of the IRE-1/XBP-1 pathway, and with an advanced set of proteasome activity-specific fluorescent affinity probes that allow direct, selective, simultaneous visualization of the activity of all six active β-subunits of the constitutive and the immunoproteasome. Our results demonstrate that the A310G mutation has a modest impact on β5c proteasome inhibition by bortezomib (increasing the IC50 from 25nM to 80 nM), and likewise by carfilzomib (IC50 increase from 10 nM to 50 nM). Strikingly, when AMO-CFZ or AMO-BTZ were exposed to the same functional level of proteasome inhibition (> 90% inhibition of β5c/5i, 20% inhibition of β1/1i, β2/2i) that resulted in 70-90% cytotoxicity in AMO-1 cells, no cytotoxicity was observed in AMO-BTZ and AMO-CFZ cells. Likewise, AMO-BTZ and AMO-CFZ cells were resistant to the next generation proteasome inhibitors ixazomib, oprozomib and dalanzomib, irrespective of the presence or absence of the PSMB5 mutation. Analysis of the UPR and its major regulators on protein and mRNA levels revealed that all clones of AMO-BTZ and AMO-CFZ showed significantly lower expression of IRE-1 and its product, spliced XBP-1, compared to AMO-1 cells, in contrast to all other major regulators of the UPR (ATF6, PERK, elF2a). Proteasome inhibitor treatment induced phosphorylation of IRE-1 and the induction of sXBP1 similarly in AMO-1, AMO-BTZ and AMO-CFZ cells, however, the induction of downstream proteins of the UPR (ATF4, PDI) was exclusively found in AMO-1 cells. Mass spectrometry-based quantitative global proteomic analysis was performed to compare AMO-1 cells with AMO-BTZ and AMO-CFZ with a cut off of at least a 50% change in abundance of differentially expressed proteins in at least 2 out of triplicate experiments. This yielded > 3500 identified individual proteins in proteasome inhibitor adapted cells, of which > 600 were differentially expressed and subsequently subjected to a protein-protein interaction (PPI) search and a Gene Ontology (GO) analysis, resulting in an average of 30 GO terms for the overexpressed proteins and 10 for downregulated species in AMO-BTZ and AMO-CFZ. Manual grouping of GO into functionally related clusters resulted in 5-6 groups that were largely concordant between AMO-BTZ and AMO-CFZ. The clusters found overexpressed in AMO-BTZ and AMO-CFZ were proteins involved in protein catabolism, redox control and protein folding. Uniform downregulation was observed for protein clusters involved in transcription/translation, differentiation, apoptosis and structural/cytoskeletal functions. The quantitatively largest group of proteins with significantly altered expression levels in AMO-BTZ/AMO-CFZ vs. AMO-1 control cells consisted of proteins involved in metabolic regulation. This big cluster comprised close to 50 % of all polypeptides with significant quantitative changes, suggesting a key role for metabolic homeostasis. The quantitatively most significantly upregulated protein in both AMO-CFZ and AMO-BTZ was NADPH dehydrogenase, the most important reducing enzyme in eukaryotic cells (4-6 x upregulated). The top individual upregulated protein in AMO-CFZ was the p-glycoprotein 1 (Pgp, 12 x upregulated), while the transcription factor IKZF3 was among the top downregulated proteins in AMO-BTZ cells (0.2 x). Our data indicate that proteasome gene mutations are not required for proteasome inhibitor resistance of myeloma cells, that proteasome inhibitor adapted myeloma cells can compensate subtotal proteasome inhibition irrespective of the type of inhibitor used, and that they have undergone complex adaptive changes in particular in proteins that regulate metabolic functions. Thus we suggest that the metabolic machinery rather than the proteasome should be explored for drug targets in myeloma cells with acquired proteasome inhibitor resistance. DisclosuresNo relevant conflicts of interest to declare.

Four phase 3 studies of the oral proteasome inhibitor (PI) ixazomib for multiple myeloma in the newly-diagnosed, relapsed/refractory, and maintenance settings: TOURMALINE-MM1, -MM2, -MM3, and -MM4

e174 PO-162 Four phase 3 studies of the oral proteasome inhibitor (PI) ixazomib for multiple myeloma in the newly-diagnosed, relapsed/refractory, and maintenance settings: TOURMALINE-MM1, -MM2, -MM3, and -MM4 J. San Miguel, P. Moreau, V. Rajkumar, A. Palumbo, T. Facon, G. Morgan, R. Orlowski, M. Cavo, H. Einsele, F. Neumann, R. Labotka, S. Lonial, P. Richardson Clinica Universidad de Navarra, Centro Investigacion Medica Aplicada (CIMA) Pamplona, Spain; University Hospital Hotel Dieu, Nantes, France; Division of Hematology, Mayo Clinic, Rochester, MN, USA; Azienda Ospedaliero-Universitaria (AOU), University of Torino, Torino, Italy; Hopital Claude-Huriez, Lille, France; University of Arkansas for Medical Sciences (UAMS), Little Rock, AR, USA; MD Anderson Cancer Center, Houston, TX, USA; Seragnoli Institute of Haematology, University School of Medicine, Bologna, Italy; Medizinische Klinik und Poliklinik II, Julius Maximilians Universitat Wurzburg, Wurzburg, Germany; Millennium Pharmaceuticals, Inc., Cambridge, MA, USA, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited; Winship Cancer Institute of Emory University, Atlanta, GA, USA; Dana-Farber Cancer Institute, Boston, MA, USA Background: Triplet regimens combining a PI with an immunomodulatory drug (IMiD) plus dexamethasone (dex) have been shown to be highly effective for the treatment of multiple myeloma (MM), resulting in improved long-term outcomes. These improved outcomes have increased the focus on extended treatment, including maintenance therapy; however, the feasibility of long-term treatment with current regimens is limited due to toxicities or the need for regular clinic visits. Ixazomib is the first oral PI to be studied in the clinic, with encouraging early-phase data. A phase 1 study showed weekly administration of single-agent ixazomib to be TOURMALINE-MM1 TOURMALINE-MM Setting Relapsed/refractory MM Newly diagnosed MM Regimen Ixazomib plus Iendex vs Iendex Ixazomib plus Iendex vs Dosing Ixazomib 4 mg or placebo on days 1 ,8, and 15 Ixazomib 4 mg or plac on days 1, 8, and 1 Len 25 mg on days 1e21 Dex. 40 mg on days 1, 3, 15, and 22 Len 25 mg on days 1 Dex. 40 mg on day 1, 8,15, and 22 28-day cycles 28-day cycles Enrollment 722 patients Estimated 701 Primary end point PFS PFS Main secondary end points OS, OS in del17p, response rate, DOR, TTP, safety, PFS and OS in patients with high-risk cytogenetics OS, response rate, DOR, TTP, safety, PFS and O patients with high-ris cytogenetics Abbreviations: ASCT, autologous stem cell transplantation; dex, dexamethasone; DOR, duration of r survival; TTP, time to progression: TTR, time to response. 15th International Myeloma Workshop, September 23-26, 2015 generally well tolerated in patients with relapsed/refractory MM, with a low incidence of peripheral neuropathy (PN) (20% overall, only 1 case of grade 3 PN), and encouraging preliminary efficacy (Kumar et al Blood 2014). The feasibility of the all-oral combination of ixazomib plus lenalidomide (len) and dex has also been demonstrated, with a 90% overall response rate (including 62% VGPR) and a manageable safety profile with limited PN (6% grade 3) reported in patients with newly diagnosed MM (Kumar et al Lancet Oncol 2014). These studies, together with population pharmacokinetic results, identified the recommended ixazomib phase 3 dose as 4 mg weekly for patients with relapsed/refractory or newly diagnosed MM (Gupta et al Br J Clin Pharmacol 2015). The recommended phase 3 dose for ixazomib maintenance therapy was identified as 3 mg, increasing to 4 mg after 4 cycles if tolerated, using an exposure-safety-efficacy analysis approach (Gupta et al EHA 2014). On the basis of these preliminary data, 4 phase 3 trials of ixazomib are ongoing in the following settings: relapsed and/or refractory MM (TOURMALINE-MM1, NCT01564537), newly diagnosed MM (NDMM) (TOURMALINE-MM2, NCT01850524), MM maintenance therapy following autologous stem cell transplantation (ASCT) (TOURMALINE-MM3, NCT02181413), MM maintenance therapy after initial therapy without ASCT (TOURMALINE-MM4, NCT02312258). Methods: The four trials are shown in the Table. All are randomized, placebo-controlled, double-blind, multicenter studies. Patients in the TOURMALINE-MM1 and -MM2 studies will receive the all-oral combination of ixazomib plus len-dex, while those in the TOURMALINE-MM3 and -MM4 maintenance trials will receive single-agent ixazomib. Progression-free survival is the primary endpoint for all four trials. Recruitment for TOURMALINEMM2, -MM3, and -MM4 is ongoing The authors would like to thank Jane Saunders of FireKite, an Ashfield company, part of UDG Healthcare plc, for writing support during the development of this abstract, which was funded by Millennium Pharmaceuticals, Inc. 2 TOURMALINE-MM3 TOURMALINE-MM4 MM maintenance post-ASCT MM maintenance without ASCT Iendex Ixazomib maintenance vs placebo Ixazomib maintenance vs placebo ebo 5 Ixazomib 3 mg or placebo on days 1, 8, and 15 for cycles 1e4 Ixazomib 3 mg or placebo on days 1, 8, and 15 for cycles 1e4 -21 s Ixazomib 4 mg or placebo on days 1, 8, and 15 for cycles 5e26 Ixazomib 3 or 4 mg, or placebo on days 1 , 8, and 15 for cycles 5e26 28-day cycles 28-day cycles Estimated 652 Estimated 761

Abstract A18: MicroRNA-29b replacement inhibits proteasomes and disrupts the aggresome-autophagy pathway to enhance the antimyeloma benefit of bortezomib.

Abstract Purpose: The goal of the present study was to identify and functionally characterize novel microRNAs (miRNAs) that inhibit the ubiquitin+proteasome system (UPS) and also disrupt the aggresome+autophagy pathway for anti-myeloma benefit. Background: Evading apoptosis is a cancer hallmark that remains a serious obstacle in current treatment approaches. Drug resistance accounts for the majority of tumor relapses and cancer-related deaths. Functional blockade of the UPS using targeted therapeutics has translated into clinical success and transformed the management of MM. However, drug resistance emerges through induction of the aggresome+autophagy pathway as a compensatory protein clearance mechanism leading to treatment failure, disease relapse and ultimately fatal outcome. Experimental Procedures: Myeloma cells were exposed to the proteasome inhibitors bortezomib, carfilzomib or ixazomib to generate cells with acquired drug resistance. Genome-wide, microarray-based profiling was performed to identify miRNAs significantly deregulated in the drug-resistant myeloma cells relative to parental drug-naïve cells. Based upon the miRNA profiling, we identified a curated panel of miRNAs that were significantly reduced in drug-resistant cells. To determine the effect of individual miRNAs, synthetic miRNA replacements were transfected into myeloma cells. Individual miRNA replacements were also transfected into human embryonic kidney (HEK) cells that expressed a short-lived green fluorescent protein that is degraded by the proteasome. The effect of individual miRNAs on the UPS was then quantitated in HEK cells by confocal microscopy and fluorescent imaging. Results: MiRNA-29b was significantly downregulated in the myeloma cells generated with acquired resistance to bortezomib, carfilzomib and ixazomib relative to the parental cells. MiRNA-29b was also downregulated in MM patient tumor cells relative to plasma cells form healthy individuals. MiRNA-29b targeted PSME4 which encodes the proteasome activator PA200. Transfection of synthetically-engineered miRNA-29b replacements reduced the viability of myeloma cells and patient tumor cells and synergistically enhanced the cytotoxic effect of proteasome inhibitors. The miRNA-29b replacement also reduced the growth of myeloma xenografts in mouse models in vivo. MiRNA-29b replacements decreased expression of PSME4 and the protein product PA200, a proteasome activator. Reduction in PA200 reduced the proteasome's chymotrypsin-like peptidase activity and inhibited ornithine decarboxylase turnover, a proteasome substrate degraded through Ub-independent mechanisms. Following proteasome inhibition, perinuclear aggregates of insoluble ubiquitinated proteins, termed aggresomes, accumulate and are degraded by the autophagy pathway. Importantly, in contrast to bortezomib, miRNA-29b replacement inhibited proteasome activity but did not induce the formation of perinuclear aggresomes or autophagosomes. Rather, immunofluorescence studies indicated that miRNA-29b replacement promoted the accumulation of small, ubiquitinated protein aggregates that were dispersed throughout the cytoplasm but were not transported by microtubules and were not coalesced into mature aggresomes. The ubiquitinated proteins also were not detected in autophagosomes. Conclusions: MiRNA-29b replacement represents the first-in-class miRNA-based agent to inhibit proteasomes and uncouple the UPS from the aggresome-autophagy pathway. Taken together, our study highlights the potential of miRNA replacement therapy to synergistically enhance the anti-myeloma effect of PIs. Citation Format: Sajjeev Jagannathan, Nikhil Vad, Mohamed Abdel-Malek, Ehsan Malek, James Driscoll. MicroRNA-29b replacement inhibits proteasomes and disrupts the aggresome-autophagy pathway to enhance the antimyeloma benefit of bortezomib. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A18.

Abstract LB-245: Systems-based pharmacogenomics reveals that the ubiquitin ligase SCF-SKP2 is a molecular determinant of clinical response to bortezomib-based therapy

Abstract The molecular events that dictate the initiation, progression and therapeutic response in multiple myeloma (MM) remain incompletely understood. The ubiquitin (Ub) ligase Skp1-Cullin-1-Skp2 (SCFSkp2) complex promotes proliferation by inducing proteasomal degradation of the cyclin-dependent kinase inhibitor p27Kip1. Skp2 overexpression and reduced p27 levels are frequent events in human cancers and are associated with poor prognosis. Methods: Prospective pharmacogenomics was performed using next generation exome sequencing gene expression profiles (GEPs) compiled from the APEX/SUMMIT phase III clinical trial deposited in dataset GSE9782. 255 relapsed MM patients were randomized to bortezomib (BTZ) vs. dexamethasone (DEX) alone and progression-free (PFS) and overall survival (OS) then correlated with the pre-treatment GEPs. RPMI8226 cells were exposed for 8 months to successively escalated doses of the proteasome inhibitors BTZ, carfilzomib or ixazomib to generate chemoresistant cell lines. Side population (SP) cells were isolated from MMCLs and patients’ samples by flow cytometry sorting based upon Hoechst 33342 staining. Results: Pharmacogenomic correlation of GEPs with clinical response revealed hyperexpression of CUL1 and SKP2 in tumors from MM patients that did not respond to BTZ. CUL1 and SKP2 hyperexpression was correlated with significantly reduced PFS and OS after treatment with BTZ but not with DEX. Cullin-1 and Skp2 were elevated in cells generated with acquired PI-resistance. CUL1 or SKP2 genetic ablation significantly enhanced the sensitivity of chemoresistant cells to PIs. A high-throughput assay was then employed using GFP-tagged p27 to screen large databases and chemical libraries in order to identify novel lead compounds that inhibited the Skp2-dependent ubiquitination of p27. Treatment with DT204, a novel lead compound identified here, reduced p27 ubiquitination, its accumulation in myeloma cells and impaired Skp2 association with Cullin-1 and Cks1, a critical rate-limiting effector of Skp2 activity. The anti-myeloma effect of DT204 was diminished using a phosphorylation-defective p27 mutant. Co-treatment with DT204 enhanced the anti-myeloma effect of BTZ against MM cell lines, patient tumor cells, stem cells and the tumor-initiating clonogenic side population (SP). The in-vivo efficacy and survival benefit of DT204 as monotherapy or in combination with BTZ will be reported. Conclusion: Taken together, the results demonstrate that pharmacologics to selectively disrupt the SCFSkp2 complex-p27 axis within the UPS holds promise to overcome chemoresistance with therapeutic benefit for myeloma patients. Skp2, Cullin1 can be utilized as biomarkers to select MM patients who will benefit from early experimental therapeutics of Skp2-inhibitors. Note: This abstract was not presented at the meeting. Citation Format: Ehsan Malek, Mohamed Abdel Malek, James J. Driscoll. Systems-based pharmacogenomics reveals that the ubiquitin ligase SCF-SKP2 is a molecular determinant of clinical response to bortezomib-based therapy. [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 LB-245. doi:10.1158/1538-7445.AM2015-LB-245

Integrated nonclinical and clinical risk assessment of the investigational proteasome inhibitor ixazomib on the QTc interval in cancer patients.

Ixazomib is the first oral, proteasome inhibitor to reach phase III trials. Here, we present an integrated nonclinical and clinical assessment of ixazomib's effect on QTc intervals. Nonclinical studies assessed (1) the in vitro binding of ixazomib to the hERG channel and (2) its effect on QT/QTc in dogs (N = 4) via telemetry. Pharmacokinetic-matched triplicate electrocardiograms were collected in four clinical phase I studies of intravenous (0.125-3.11 mg/m(2), N = 125, solid tumors/lymphoma) or oral (0.24-3.95 mg/m(2), N = 120, multiple myeloma) ixazomib. The relationship between ixazomib plasma concentration and heart rate (HR)-corrected QT using Fridericia (QTcF) or population (QTcP) methods was analyzed using linear mixed-effects models with fixed effects for day and time. In vitro binding potency for ixazomib to the hERG channel was weak (K i 24.9 μM; IC50 59.6 μM), and nonclinical telemetry studies showed no QT/QTc prolongation at doses up to 4.2 mg/m(2). In cancer patients, ixazomib, when evaluated at doses yielding various plasma concentrations (with 26 % of data greater than mean C max for the 4 mg phase 3 dose), had no meaningful effect on QTc based on model-predicted mean change in QTcF/QTcP from baseline. There was no relationship between ixazomib concentration and RR, suggesting no effect on HR. Ixazomib has no clinically meaningful effects on QTc or HR. Integrating preclinical data and concentration-QTc modeling of phase 1 data may obviate the need for a dedicated QTc study in oncology. A framework for QT assessment in oncology drug development is proposed.

Switching from body surface area-based to fixed dosing for the investigational proteasome inhibitor ixazomib: a population pharmacokinetic analysis.

This population pharmacokinetic analysis of the investigational oral proteasome inhibitor ixazomib assessed the feasibility of switching from body surface area (BSA)-based to fixed dosing, and the impact of baseline covariates on ixazomib pharmacokinetics. Data were pooled from 226 adult patients with multiple myeloma, lymphoma or solid tumours in four phase 1 studies, in which ixazomib dosing (oral/intravenous, once/twice weekly) was based on BSA. Population pharmacokinetic modelling was undertaken using nonmem version 7.2. Ixazomib pharmacokinetics were well described by a three compartment model with first order absorption and linear elimination. Ixazomib was absorbed rapidly (Ka 0.5 h(-1)), with dose- and time-independent pharmacokinetics. Estimated absolute bioavailability and clearance were 60% and 2l h(-1), respectively. Although a small effect of BSA (range 1.3-2.6 m(2)) was observed on the peripheral volume of distribution (V4), reducing the corresponding inter-individual variability by 12.9%, there was no relationship between BSA and ixazomib clearance (the parameter that dictates total systemic exposure following fixed dosing). Consistently, based on simulations (n = 1000), median AUCs (including interquartile range) were similar after BSA-based (2.23 mg m(-2)) and fixed (4 mg) oral dosing with no trend in simulated AUC vs. BSA for fixed dosing (P = 0.42). No other covariates, including creatinine clearance (22-213.7 ml min(-1)) and age (23-86 years), influenced ixazomib pharmacokinetics. This analysis supports a switch from BSA-based to fixed dosing, without dose modification for mild/moderate renal impairment or age, in future adult studies of ixazomib, simplifying dosing guidance and clinical development.

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.

CCR 20th anniversary commentary: In the beginning, there was PS-341.

Proteasome inhibitors have a 20-year history in cancer therapy. The first proteasome inhibitor, bortezomib, a breakthrough treatment for multiple myeloma, moved rapidly through development from the bench in 1994 to first FDA approval in 2003. Clinical Cancer Research has chronicled the development of proteasome inhibitors with publication of reports on bortezomib, carfilzomib, and the oral proteasome inhibitor ixazomib (MLN9708).

Transitioning to an All Oral Therapy for Multiple Myeloma with the Novel Proteasome Inhibitor Ixazomib

Transitioning to an All Oral Therapy for Multiple Myeloma with the Novel Proteasome Inhibitor Ixazomib

Long-Term Outcome of a Phase 1 Study of the Investigational Oral Proteasome Inhibitor (PI) Ixazomib at the Recommended Phase 3 Dose (RP3D) in Patients (Pts) with Relapsed or Refractory Systemic Light-Chain (AL) Amyloidosis (RRAL)

Long-Term Outcome of a Phase 1 Study of the Investigational Oral Proteasome Inhibitor (PI) Ixazomib at the Recommended Phase 3 Dose (RP3D) in Patients (Pts) with Relapsed or Refractory Systemic Light-Chain (AL) Amyloidosis (RRAL)

Phase 1 Study of the Investigational Proteasome Inhibitor Ixazomib Alone or in Combination with Lenalidomide–Dexamethasone (Rd) in Japanese Patients (Pts) with Relapsed and/or Refractory Multiple Myeloma (RRMM)

Background Ixazomib is the first oral proteasome inhibitor to be investigated clinically for the treatment of MM. Phase 1 studies have shown single-agent activity and manageable toxicities in RRMM (Kumar et al. Blood 2014) and phase 1/2 studies have suggested the feasibility and activity of weekly oral ixazomib plus Rd in previously untreated MM (Kumar et al. ASH 2012; Richardson et al. ASH 2013). These findings have led to ongoing phase 3 trials of weekly ixazomib 4 mg + Rd in RRMM and previously untreated MM. However, the early-phase studies were conducted in Western pts. This phase 1, open-label multicenter study aimed to determine the safety, tolerability, and pharmacokinetics (PK) of weekly ixazomib alone or with Rd in Japanese pts with RRMM (Japic Clinical Trials Information no. 121822). Methods Primary objectives were to evaluate the safety and tolerability, including dose-limiting toxicities (DLTs) and adverse events (AEs), and the PK of ixazomib alone or with Rd. A secondary objective was evaluation of antitumor activity. Japanese pts aged ≥20 years with RRMM who had received at least 2 prior regimens, which must have included bortezomib, thalidomide or lenalidomide, and corticosteroids, were eligible. All had measurable disease and ECOG performance status of 0–2. Pts with grade ≥2 peripheral neuropathy or grade ≥2 diarrhea at study entry were excluded. Pts received ixazomib 4 mg on days 1, 8, and 15 of 28-day cycles, alone or with Rd (lenalidomide 25 mg on days 1–21, dexamethasone 40 mg on days 1, 8, 15, and 22), per the regimen used in the ongoing phase 3 trials. AEs were graded per NCI-CTCAE v4.03. Blood samples for PK analysis were taken at multiple time points prior to and after dosing on days 1 and 15 of cycle 1. Responses were assessed per IMWG uniform response criteria. Results Fourteen pts were enrolled; 8 (57%) were male, median age was 62.5 yrs (range 53–71), 4 pts were aged ≥65 yrs, median number of prior therapies was 7. Seven pts received single-agent ixazomib and 7 received ixazomib + Rd. One pt in each cohort was excluded from the DLT-evaluable population. Two patients experienced DLTs in cycle 1: 1 pt receiving single-agent ixazomib had grade 4 thrombocytopenia and grade 3 diarrhea, hypertension, hypokalemia, hyponatremia, and nausea; 1 pt in the ixazomib + Rd cohort had grade 4 thrombocytopenia and neutropenia. All events were considered treatment-related. At data cut-off (Jan 6 2014), 6 pts remained on treatment and 8 had discontinued due to: progressive disease (PD; n=3), AEs (n=3), symptomatic deterioration, and protocol violation (each n=1). At data cut-off, pts (n=14) had received a median of 6 cycles of ixazomib (range 1–21); the 7 pts in the ixazomib + Rd cohort had received a median of 4 cycles (range 1–12) of ixazomib + Rd. Thirteen (93%) pts experienced treatment-related AEs; the most common were neutropenia (71%), thrombocytopenia (71%), leukopenia (64%), lymphopenia (57%), and diarrhea (50%). There were no cases of peripheral neuropathy. Nine (64%) pts had grade ≥3 AEs; the most common were lymphopenia (50%), neutropenia (43%), and thrombocytopenia (36%). Two (14%) pts (single-agent cohort) had serious AEs (grade 2 bronchitis in 1 pt, and grade 4 thrombocytopenia and grade 3 hypokalemia in 1 pt). Three pts discontinued due to AEs; 1 due to diarrhea in the single-agent cohort, and 1 due to neutropenia and 1 due to thrombocytopenia in the ixazomib + Rd cohort. There were no deaths. PK data showed ixazomib was rapidly absorbed with a Tmax at 1.08–1.83 hrs. Terminal half-life (geometric mean) was 5.7 days for single-agent ixazomib and 5.2 days for ixazomib + Rd. There were no substantial differences in the ixazomib PK profile between the two cohorts. Thirteen pts were response-evaluable. One pt (ixazomib + Rd cohort) had a partial response; at data cut-off, this pt remained in response with a 100% M-protein reduction (unconfirmed VGPR) and duration of response of ~10.8 months. Seven pts had stable disease (including 3 with M-protein reductions of 25–50%), 2 had PD, and 3 were not assessable. Conclusions These data suggest that ixazomib 4 mg alone or with Rd is feasible and tolerable in Japanese pts with RRMM. The AEs were manageable, reflecting the AE profile seen in Western populations, supporting the use of this dose and schedule in Japanese pts. Disclosures Handa: Celgene: Research Funding; Yakult: Research Funding; Kirin: Research Funding; Chugai: Research Funding. Off Label Use: Investigational agent ixazomib for the treatment of Japanese patients with relapsed and/or refractory multiple myeloma.. Matsushima:Takeda Pharmaceutical Company Limited : Employment.

Long-Term Ixazomib Maintenance Is Tolerable and Improves Depth of Response Following Ixazomib-Lenalidomide-Dexamethasone Induction in Patients (Pts) with Previously Untreated Multiple Myeloma (MM): Phase 2 Study Results

Long-Term Ixazomib Maintenance Is Tolerable and Improves Depth of Response Following Ixazomib-Lenalidomide-Dexamethasone Induction in Patients (Pts) with Previously Untreated Multiple Myeloma (MM): Phase 2 Study Results

Generation of a Predictive Score for Ixazomib Response in Multiple Myeloma

Multiple myeloma (MM) is the second-most common hematopoietic malignancy in the United States accounting for 1% of all cancers and 10% of all hematologic malignancies. Despite recent improvements in treatment strategies including the emergence of proteasome inhibitors (PIs) as effective chemotherapeutic agents, MM still remains difficult to cure with median survival rate of around 7 years, primarily due to wide inter-individual variation in response to treatment. We believe such heterogeneity in response to PIs is governed by the underlying molecular characteristics of the tumor including alterations in gene expression profile (GEP).In the current study, we used a panel of Human Myeloma Cell Lines (HMCLs) representing the gamut of biological and genetic heterogeneity in MM to evaluate the gene expression signatures associated with response to the second-generation PI Ixazomib and produced a predictive score (PI score) for Ix response.HMCLs (n=45) were treated with increasing concentrations of Ixazomib used as single agent and half maximal inhibitory concentration (IC50) values were determined using cell viability equation. Gene expression profiling data was obtained as publicly available data from the Keats lab website at TGen (http://www.keatslab.org/myeloma-cell-lines).Genes with high expression value and high standard deviation beyond the median values were pre-filtered and log expression values were normalized by subtracting mean expression of individual genes across all the samples and the housekeeping genes (GAPDH). Subsequently, analysis of correlation between Ix IC50 data and GEP data and the False Discovery Rate (FDR) based on 1000 random permutations were performed to identify true patterns of genes that are highly predictive of Ix response and to look for the top genes that could discriminate between the top sensitive and top resistant cell lines. Gene clusters were identified that correlated with response and will be presented. Our results will demonstrate in vitro modeling of response using GEP approaches that may provide predictive scoring algorithms of a defined set of genes that will be useful in clinical evaluation of drug choice in treating individual patients. DisclosuresNo relevant conflicts of interest to declare.

Phase 1 study of weekly dosing with the investigational oral proteasome inhibitor ixazomib in relapsed/refractory multiple myeloma

AbstractProteasome inhibition is an effective treatment strategy for multiple myeloma. With improving survival, attention is increasingly focusing on ease of administration and toxicity profile. Ixazomib is an investigational, orally bioavailable 20S proteasome inhibitor. Sixty patients with relapsed and/or refractory multiple myeloma were enrolled on this phase 1 trial to evaluate safety and tolerability and determine the maximum tolerated dose (MTD) of single-agent, oral ixazomib given weekly for 3 of 4 weeks. Upon MTD determination, patients were enrolled to 4 different cohorts based on relapsed/refractory status and prior bortezomib and carfilzomib exposure. The MTD was determined to be 2.97 mg/m2. Dose-limiting toxicities were grade 3 nausea, vomiting, and diarrhea in 2 patients, and grade 3 skin rash in 1 patient. Common drug-related adverse events were thrombocytopenia (43%), diarrhea (38%), nausea (38%), fatigue (37%), and vomiting (35%). The observed rate of peripheral neuropathy was 20%, with only 1 grade 3 event reported. Nine (18%) patients achieved a partial response or better, including 8 of 30 (27%) evaluable patients treated at the MTD. Pharmacokinetic studies suggested a long terminal half-life of 3.6 to 11.3 days, supporting once-weekly dosing. This trial was registered at www.clinicaltrials.gov as #NCT00963820.