Proteasome Inhibitor Ixazomib Research Articles

Glutathione-responsive copper-disulfiram nanoparticles for enhanced tumor chemotherapy

Disulfiram (DSF), a familiar FDA-approved drug used for alcohol withdrawal, has recently been verified with potent antitumor therapeutic effect by generating Cu(DTC)2, which is the complex of its metabolite diethyldithiocarbamate (DTC) and copper. However, its poor tumor selectivity and insufficient endogenous Cu2+ concentration within tumor site largely hinders the application of DSF-based antitumor therapy. Therefore, a GSH-responsive coordination nanoparticles (Cu-IXZ@DSF) was established as a copper carrier to achieve synchronous but separate delivery of Cu2+ and DSF without antitumor ability, further to realize selectively triggered tumor in situ Cu(DTC)2 generation for antitumor therapy. A widely-used proteasome inhibitor ixazomib (IXZ) was chosen as ligands and Cu2+ was used as coordination nodes to form nanosized Cu-IXZ@DSF. The DSF encapsulated in Cu-IXZ@DSF could be reduced to DTC by intracellular GSH, which could contend for Cu2+ and realize in situ high toxic Cu(DTC)2 generation. Meanwhile, the chelation could lead to the disassembly of Cu-IXZ@DSF and release of IXZ to eventually achieve tumor specific “transformation from low toxicity to high toxicity” chemotherapy. The results of in vitro and in vivo experiments demonstrated that the as-prepared nanoplatform Cu-IXZ@DSF showed good biosafety and excellent antitumor effect via endoplasmic reticulum stress (ERS) as well as reactive oxygen species (ROS) generation pathway. Therefore, this nanocarrier provides an inspiring strategy with specific-triggered antitumor Cu(DTC)2 generation for DSF-based chemotherapy with high therapeutic effect and biosafety and showing great potential of treating cancer.

Lenalidomide Compared to Ixazomib Maintenance in Newly Diagnosed Multiple Myeloma: A Systematic Review and Meta-Analysis

BackgroundMaintenance therapy is considered a standard of care in transplant eligible (TE) and non transplant eligible (NTE) patients with newly diagnosed multiple myeloma (NDMM). While immunomodulators (IMID) and proteasome inhibitors (PI) have been proposed as maintenance therapy options, there are no randomised trials (RCTs) directly comparing these agents in the maintenance setting. The IMID lenalidomide (Len) and the PI ixazomib (Ixa) have been compared against placebo as maintenance strategies in NDMM. We present a network meta-analysis (NMA) of RCTs comparing the efficacy and safety of Len and Ixa maintenance therapies in NDMM.MethodsWe searched various biomedical databases for eligible studies evaluating Len or Ixa against placebo/ observation as maintenance therapy in patients with NDMM from date of inception through November 2020. The primary outcome was progression-free survival (PFS). Secondary outcomes include overall survival (OS) and adverse events (AE). The Cochrane risk of bias tool version 2.0 was used to assess trial quality. A Bayesian NMA model was used to assess the relative effects of competing treatments on PFS and OS outcomes. Adverse events were assessed using the synthesis without meta-analysis (SWIM) approach due to variability in the toxicity scoring criteria. The GRADE approach was used to rate the certainty of the evidence. This study is registered with PROSPERO, CRD42021226157.ResultsWe identified eight studies including 3229 transplant eligible (TE) and 1689 non transplant eligible (NTE) patients. All studies were judged to have low risk of bias. Len but not Ixa was associated with statistically significant improvement in PFS when compared to placebo in TE (Len: Hazard Ratio (HR) 0.46, 95% Credible Interval (CrI) 0.35-0.56, high certainty; Ixa: HR 0.72, 95% CrI 0.46-1.13, moderate certainty) and NTE (Len: HR 0.46, 95% CrI 0.29-0.75, high certainty; Ixa: HR 0.69, 95% CrI 0.43-1.18, moderate certainty). Bayesian modelling demonstrated a 97% and 93% probability that Len resulted in superior PFS compared to Ixa in TE and NTE patients respectively. Both Len and Ixa were not associated with statistically significant improvement in OS compared to placebo in TE and NTE patients. There was no significant effect modification on the effect of Len vs placebo and Ixa vs placebo by cytogenetics status, use of proteasome inhibitors for induction or duration of maintenance therapies for PFS and/or OS outcomes. Len were judged to have a higher incidence of second malignancies and grade 3 or 4 neutropenia than Ixa while Ixa was judged to have a higher incidence of thrombocytopenia than Len.ConclusionsMaintenance therapy with Len may provide a larger PFS benefit than Ixa regardless of type of induction therapy and cytogenetic risk in patients with NDDM. The differing toxicity profile of these agents is also an important consideration for treatment decisions. RCTs directly comparing these maintenance strategies are warranted. [Display omitted] DisclosuresOoi: Jansen: Honoraria; Teva Pharmaceuticals: Honoraria; GSK: Honoraria; Abbvie: Honoraria; Amgen: Honoraria. Chng: Sanofi: Honoraria; Abbvie: Honoraria; Takeda: Honoraria; Amgen: Honoraria; BMS/Celgene: Honoraria, Research Funding; Johnson and Johnson: Honoraria, Research Funding; Pfizer: Honoraria.

Late vs Early Response and Depth of Response Are Associated with Improved Outcomes in Newly Diagnosed Multiple Myeloma (NDMM) Patients (pts) Treated with Ixazomib-Lenalidomide-Dexamethasone (IRd) or Placebo-Lenalidomide-Dexamethasone (pbo-Rd) in the Phase 3 TOURMALINE-MM2 Trial

Late vs Early Response and Depth of Response Are Associated with Improved Outcomes in Newly Diagnosed Multiple Myeloma (NDMM) Patients (pts) Treated with Ixazomib-Lenalidomide-Dexamethasone (IRd) or Placebo-Lenalidomide-Dexamethasone (pbo-Rd) in the Phase 3 TOURMALINE-MM2 Trial

Interim Analyses of Overall Survival (OS) from the TOURMALINE MM3 & MM4 Studies of Ixazomib Maintenance Following Primary Therapy in Multiple Myeloma (MM)

Interim Analyses of Overall Survival (OS) from the TOURMALINE MM3 & MM4 Studies of Ixazomib Maintenance Following Primary Therapy in Multiple Myeloma (MM)

Phase II Trial of Ixazomib and Dexamethasone Versus Ixazomib, Dexamethasone and Lenalidomide, Randomized with NFKB2 Rearrangement. (Proteasome Inhibitor NFKB2 Rearrangement Driven Trial, PINR)

Phase II Trial of Ixazomib and Dexamethasone Versus Ixazomib, Dexamethasone and Lenalidomide, Randomized with NFKB2 Rearrangement. (Proteasome Inhibitor NFKB2 Rearrangement Driven Trial, PINR)

Extended Characterization of Newly Diagnosed Multiple Myeloma (NDMM) Patients with In-Class Transition (iCT) from Parenteral Bortezomib to Oral Ixazomib Proteasome Inhibitor (PI) Therapy in the Community-Based United States (US) MM-6 Study: Updated Efficacy and Safety, and Reasons for Premature Discontinuation

Extended Characterization of Newly Diagnosed Multiple Myeloma (NDMM) Patients with In-Class Transition (iCT) from Parenteral Bortezomib to Oral Ixazomib Proteasome Inhibitor (PI) Therapy in the Community-Based United States (US) MM-6 Study: Updated Efficacy and Safety, and Reasons for Premature Discontinuation

Progression-Free Survival (PFS) According to the Presence of Adverse Cytogenetic Abnormalities in Patients (pts) with Multiple Myeloma (MM) Receiving Ixazomib (ixa)-Based vs Placebo (pbo)-Based Therapy: A Pooled Analysis of the TOURMALINE-MM1, MM2, MM3, and MM4 Phase 3 Studies

Progression-Free Survival (PFS) According to the Presence of Adverse Cytogenetic Abnormalities in Patients (pts) with Multiple Myeloma (MM) Receiving Ixazomib (ixa)-Based vs Placebo (pbo)-Based Therapy: A Pooled Analysis of the TOURMALINE-MM1, MM2, MM3, and MM4 Phase 3 Studies

Delayed administration of ixazomib modifies the immune response and prevents chronic graft-versus-host disease

In this study, we aimed to modify the immune response in the long term after allogeneic bone marrow transplantation (allo-BMT) by using the proteasome inhibitor ixazomib (IXZ) at the late stages of the post-transplant period. This approach facilitated the immune reconstitution after transplantation. IXZ significantly prolonged survival and decreased the risk of chronic graft-versus-host disease (cGvHD) in two different murine models without hampering the graft-versus-leukemia (GvL) effect, as confirmed by bioluminescence assays. Remarkably, the use of IXZ was related to an increase of regulatory T cells both in peripheral blood and in the GvHD target organs and a decrease of effector donor T cells. Regarding B cells, IXZ treated mice had faster recovery of B cells in PB and of pre-pro-B cells in the bone marrow. Mice receiving ixazomib had a lower number of neutrophils in the GvHD target organs as compared to the vehicle group. In summary, delayed administration of IXZ ameliorated cGvHD while preserving GvL and promoted a pro-tolerogenic immune response after allo-BMT.

Targeting lipid metabolism in multiple myeloma cells: Rational development of a synergistic strategy with proteasome inhibitors.

Aberrant lipid metabolism is recognized as a key feature of cancer cells. Our initial research on MS-based analysis of lipids in a multiple myeloma (MM) cell line showed a significant accumulation of lipids in multiple myeloma cells after proteasome inhibition. This finding prompted us to hypothesize that multiple myeloma cell survival depends on the maximal utilization of abnormally accumulated lipids. Therefore, we explored whether lipid metabolism-modulating agents would synergize with proteasome inhibitors. Lipid accumulation in multiple myeloma cells was measured by MS. Synergism between lipid regulators and proteasome inhibitors was assessed by cell viability and apoptosis. A novel stable derivative of fenofibrate (FCE) was synthesized and used to treat multiple myeloma cells in vitro and in vivo along with the proteasome inhibitor ixazomib. ChIP-seq, western blotting and RT-qPCR were performed to explore the potential mechanism(s) underlying the increase in lipid levels in multiple myeloma cells after proteasome inhibition. Accumulation of lipids in multiple myeloma cells was induced by proteasome inhibition. Lipid-lowering drugs and MG-132 exerted a synergistic effect to kill multiple myeloma cells. FCE showed significant synergistic activity in vitro and in vivo with ixazomib. The abnormal lipid accumulation in multiple myeloma cells that was enhanced by proteasome inhibitors might be due to the elevated SREBP1/2 expression induced by ATF4. Our results provide a proof of principle and support for the further clinical evaluation of the combination of lipid-modulating drugs with proteasome inhibitors in the treatment of multiple myeloma.

Long-time progression-free survival in relapsed, refractory multiple myeloma with the oral ixazomib-lenalidomide-dexamethasone regime

Despite great advances in therapy, multiple myeloma is still a largely incurable disease, therefore the importance of salvage therapies is paramount. The first oral proteasome inhibitor ixazomib in combination with lenalidomide-dexamethasone is a tolerable, orally administered regime, which has become available for Hungarian relapsed, refractory multiple myeloma patients from April 2015 in the Named Patient Program. Our goal was to investigate the long-time progression-free surviving patient population treated with the ixazomib-lenalidomide-dexamethasone triplet. We retrospectively studied a total of 80 patients from 7 centers who received the triplet combination. Survival analyses were performed. Two-third of the patients responded: the overall response rate was 63.75%. 14 patients (17.5%) did not respond/had stable disease and 15 patients (18.75%) outright progressed upon therapy. Although progression-free survival was only 10.6 months for the entire patient cohort, the disease in a subgroup of 16 patients did not progress within two years. In fact, 11 of them were still in sustained remission after 3 years of therapy. Our goal was to analyze the characteristics of this subgroup. The idea of long-term therapy of multiple myeloma is gaining widespread acceptance. Therefore it is important to know which patients may benefit the most from certain therapies. Among these 16 long-term responder patients, reciprocal translocation of the immunoglobulin heavy chain seemed to lack an adverse impact on progression-free survival; comparable to trisomies, both curves had a progression-free plateau. The International Staging System (ISS) score at the start of therapy did not predict long-term survivorship. Most of the side effects in this subgroup were mild, manageable, none led to therapy discontinuation. Ixazomib-lenalidomide-dexamethasone was confirmed to be an effective and safe combination for relapsed, refractory multiple myeloma, and one-sixth of the treated patients were able to receive it for several years, effectively. This fully oral therapeutic option is at its best during the present COVID-19 pandemic. Orv Hetil. 2021; 162(36): 1451-1458.

Leflunomide Inhibits c-Myc Signaling and Impairs Myeloma Cell Growth

Introduction:Despite recent advances in treatment that have improved the prognosis for patients with multiple myeloma (MM), the disease remains incurable. There is a need for MM treatments with new mechanisms of action. Leflunomide (Lef), a commercially available oral immunosuppressive agent that has been FDA-approved for treatment of rheumatoid arthritis was evaluated as a potential MM therapeutic. In vitro anti-MM activity of Lef has previously been reported; however, the molecular mechanisms of action of Lef in MM are incompletely understood. Inhibition of Src kinase-family members and blocking of dihydroorotate dehydrogenase, an enzyme involved in pyrimidine synthesis are among the few known targets of Lef. To identify novel direct targets of Lef and to further our understanding of Lefs' mode of action on MM cells, we performed in vitro kinase screening assays and RNA/miRNA sequencing analysis of cells treated with or without Lef.Methods and Results:Our pre-clinical studies with teriflunomide (Ter), the active metabolite of Lef, showed that it inhibited growth of MM cell lines (MM.1S, MM.1R, U266, H929, RPMI-8226, OPM-2, L-363) and primary MM patients' (CD138+) plasma cells at IC50s ranging from 30 uM to 140 uM. Pharmacokinetics analysis accompanying a phase 1 dose-escalation clinical trial in MM at our institute has demonstrated that Ter serum concentrations of 500 uM can be obtained. We also found that Ter induces cell-cycle arrest in both, glucocorticoid-sensitive (MM.1S) and resistant (MM.1R) MM cell lines at <150 uM. At higher in vitro concentrations (100 uM to 200 uM), Ter induces apoptosis in MM cells. Moreover, Ter synergized with dexamethasone as well as lenalidomide in in vitro growth inhibition of MM cell line MM.1S. In vivo mouse studies with Lef as single agent or in combination with the immune modulatory drug lenalidomide or with the proteasome inhibitor ixazomib are currently ongoing and results will be presented at the meeting. To identify novel direct targets of Ter, a screening assay described by Anastassiadis et al. (PMCID: PMC3230241) was applied to test the inhibitory activity of Ter on 600 known kinases. We identified 55 kinases that were inhibited >50% at 200 uM Ter. Subsequent dose-response kinase assays identified, among others, Aurora A and PIM family members, all of which are involved in MM pathogenesis, as novel direct targets of Lef, with IC50s ranging from 35 uM to 53 uM. To identify MM-associated mRNAs and miRNAs whose expression levels are frequently altered upon Lef exposure, 4 MM cell lines (RPMI-8226, U266, MM.1S, NCI-H929) and CD138-enriched primary MM cells were exposed to 200 µM Ter or DMSO control for 24h before extraction, purification and sequencing of mRNA and microRNA. RNA-sequencing analysis and DAVID functional annotation revealed that similar changes were present between patient samples and cell lines. Pathways that were predominantly affected by Ter included purine/pyrimidine metabolism, DNA-replication and cell-cycle regulation. Gene set enrichment analysis revealed that c-Myc, an oncogene that contributes to the malignant phenotype of MM and a known promising target in MM, signaling was impaired upon Ter treatment, in MM.1S cells. Differential expression analysis, real-time PCR and western blot analysis showed that c-Myc mRNA, c-Myc protein and c-Myc-regulated miRNAs belonging to the oncomir-1 cluster miR-17-92 were all down-regulated upon Ter exposure in the majority of the MM cells. Since PIM family members are involved in c-Myc signaling in cancer cells, we performed in silico molecular docking studies using Ter/PIM. Our results suggest that the active metabolite of Lef, Ter, can directly bind to the PIM family of proteins by binding to the ATP-binding site, thereby inhibiting its kinase activity. Since pan-PIM inhibitors such as PIM447 have been reported to be cytotoxic for myeloma cells due to decrease in c-Myc levels, these results suggest that decrease in c-Myc induced by Ter may be due to PIM inhibition.Conclusion:Leflunomide impairs in vitro MM cell growth - at least in part - through inhibition of c-Myc signaling. A clinical trial with leflunomide in MM has been initiated and a separate abstract with encouraging initial results has been submitted for presentation at this meeting. DisclosuresKrishnan:Onyx: Speakers Bureau; Sutro: Consultancy; Janssen: Consultancy, Speakers Bureau; Takeda: Speakers Bureau; Celgene: Consultancy, Equity Ownership, Speakers Bureau. Rosenzweig:Celgene: Speakers Bureau.

Validation of FOXM1 As a Therapeutic Target in Acute Myeloid Leukemia

Objectives:Forkhead box M1 (FOXM1) is a transcription factor of the Forkhead family that induces genes critical in executing the mitotic program. The FOXM1 regulatory network is a major predictor of adverse survival outcomes across 18000 human tumor expression signatures (Gentles. Nat Med 2015). FOXM1 interacts with nucleophosmin (NPM) in cancer cells and NPM determines the cellular localization of FOXM1. In the OCI-AML3 cell line and in AML primary samples with mutant alleles of NPM1, we have shown that FOXM1 and mutant NPM are re-localized to the cytoplasm (Khan. Leukemia 2017). We postulated that nuclear export of FOXM1 may underlie the chemotherapy responsiveness in NPM1mut patients. We conducted a multi-institution study to investigate the nuclear expression level of FOXM1 as a predictor of chemotherapy resistance in AML and validate it as a therapeutic target.Methods:Adult patients diagnosed with intermediate-risk AML were identified at Northwestern Memorial and the University of Illinois Hospitals. A total of 102 patients were included in the study with equivalent representation from each institution. Clinical data were collected by chart review, and corresponding bone marrow biopsy samples were retrieved under an IRB approved protocol. Tissue sections were stained with a FOXM1 antibody and scanned images were analyzed using HALO 2.0 software. All biopsy specimens were reviewed by a hematopathologist for adequacy. The percentage of total nuclei expressing FOXM1, nuclear FOXM1 intensity (optical density), and the FOXM1 nuclear:cytoplasmic ratio were quantified. Statistical analysis was performed by the Design and Analysis Core, UIC.Results:Patient characteristics are summarized in Table 1. Out of 102 patients, 77 (75%) achieved a complete remission with or without count recovery (CR/CRi). We used lines of induction therapy needed to achieve remission as a measure of chemotherapy resistance. Patients were stratified as needing 1 or >1 line of induction therapy. Induction therapy included cytarabine and anthracycline in 82 cases and hypomethylator in 19 cases.We found that patients needing >1 line of induction therapy had substantially higher levels of nuclear FOXM1 in their diagnostic bone marrow biopsy compared to patients who responded to first-line therapy (20.4% vs. 11.4% positive nuclei; p= 0.066). In logistic regression analysis, the percentage of FOXM1 positive nuclei (OR 1.85 for 10% increase in positive nuclei, p=0.02) and average nuclear intensity (OR 2.18 for 0.1 U increase in OD, p=0.05) were able to predict resistance to first-line chemotherapy. Due to variation in post-induction therapies, the institutions were considered separately for survival analysis. Nuc:Cyto FOXM1 ratio was able to predict inferior overall survival in a single institution cohort analyzed (n=45) in a model incorporating standard clinicopathologic prognostic factors. Multivariate regression analysis is shown in Table 2.We then proceeded to validate nuclear FOXM1 as a therapeutic target in AML. Short hairpin knockdown of FOXM1 in AML cell lines results in decreased colony size and number (KG-1 shFOXM1: 0.35 normalized to control, p=0.01; MV4-11 shFOXM1: 0.61, p=0.03). We have shown that proteasome inhibitors stabilize HSP70 which is a negative regulator of FOXM1 (Halasi. JBC 2016). The novel proteasome inhibitor ixazomib was tested for in vitro effect on FOXM1 in AML cells. Ixazomib inhibits the transcriptional activity of FOXM1 using a luciferase reporter cell line with inducible FOXM1. Treatment with ixazomib significantly decreases FOXM1 mRNA and inhibits FOXM1 protein expression in AML cell lines and patient samples (n=12) resulting in increased apoptosis by caspase cleavage. This is consistent with the FOXM1 auto-regulation loop (Halasi. Cell Cycle 2009). The therapeutic efficacy of ixazomib in AML was then tested in vivo. Single agent treatment with ixazomib compared to vehicle in an murine xenograft model of AML showed significant reduction in tumor volume after 3 weeks of treatment (0.89cm3 vs 1.62cm3, n=8 per group) and downregulation of FOXM1 expression.Conclusion:Our work shows for the first time that nuclear FOXM1 confers chemotherapy resistance in a clinically homogenous group of intermediate risk AML patients. Moreover we show that ixazomib, a well-tolerated novel proteasome inhibitors has therapeutic efficacy in AML models that correlates with suppression of FOXM1. [Display omitted] DisclosuresKhan:Teva: Speakers Bureau; Takeda: Research Funding; Gilead: Consultancy.

Ixazomib-Thalidomide-Low Dose Dexamethasone (ITd) Induction Followed By Maintenance Therapy with Ixazomib or Placebo in Newly Diagnosed Multiple Myeloma Patients Not Eligible for Autologous Stem Cell Transplantation; Initial Results from the Randomized Phase II HOVON-126/Nmsg 21.13 Trial

IntroductionA triplet combination including a proteasome inhibitor (PI) and an IMiD has shown significant efficacy in the setting of newly diagnosed multiple myeloma (NDMM). In addition, a role for maintenance therapy with the PI bortezomib has been suggested in non-head to head comparisons. However, in elderly patients especially non-hematological toxicity is a concern and an oral drug is preferred as maintenance therapy. Therefore, we investigated the efficacy and feasibility of an oral regimen including induction therapy with the PI ixazomib in combination with thalidomide, followed by a randomization between maintenance therapy with either ixazomib or placebo in elderly non-transplant eligible (nte) NDMM. We here report the efficacy and toxicity of the induction therapy of the first 120 patients, with subgroup analyses based on frailty and cytogenetic risk. The study is currently ongoing and results with longer follow up and the maintenance part will be reported at future meetings. This trial was registered at www.trialregister.nl as NTR4910.MethodsIn this prospective multicenter phase II trial nte-NDMM patients were treated with nine 28 day-cycles consisting of ixazomib 4mg (day 1, 8, 15), thalidomide 100 mg (day 1-28) and dexamethasone 40 mg (day 1, 8, 15, 22) followed by randomisation between either ixazomib or placebo (both day 1, 8, 15 every 28 days) until progression. Primary objectives were comparison of progression free survival (PFS) between maintenance therapy with ixazomib or placebo and to determine the overall response rate of induction therapy.Frailty was assessed by a modification of the IMWG frailty index based on age, the Charlson Comorbidity Index (retrospectively retrieved from the list of comorbidities that were present at entry) and the WHO performance as a proxy for (instrumental) Activities of Daily Living (scoring WHO 0 as 0 points, WHO 1 as 1 point, and WHO 2-3 as 2 points).High risk cytogenetics was defined as del17p, t(4;14) or t(14;16).ResultsPatient characteristics are presented in table 1. The median follow up is 17.0 months (range 0.9-28.1 months). Following induction treatment overall response rate (ORR, i.e. ≥PR) was 81% (95% confidence interval (CI) 73-87%), 44% (95% CI 35-54%) of patients reached ≥ VGPR and 10% (95% CI 5-17%) ≥ CR. ORR was 85% in patients ≤75 years versus 73% in ≥76 years (p=0.15), while for ≥VGPR these were 45% vs 42% (p=0.85) and for ≥CR 13% vs 4% (p=0.21). In responding patients, the median time to response was 1.1 month.Using the revised IMWG Frailty Index 47% of patients were frail, 28% unfit and 21% fit (4% unknown). Frailty status did not affect the efficacy of induction therapy with ITd with ORR 75%, ≥ VGPR 43% and ≥ CR 9% in frail versus 85%, 53% and 9% in unfit and 88%, 36% and 16% in fit patients (p=0.34, p=0.43 and p=0.62, respectively).At the time of this analysis cytogenetic risk could be determined in 77/120 (64%) patients, with 23% of high risk patients. Response rates were comparable in high versus standard risk patients: ORR 14/18 (78%) versus 50/59 (85%), ≥ VGPR 7/18 (39%) versus 25/59 (42%) and ≥ CR 1/18 (6%) and 4/59 (7%) (p=0.49, p=1.0 and p=1.0).Toxicity is presented in table 2. The incidence of severe neuropathy was low; 3% grade 3 and no grade 4. During induction 18/120 (15%) patients discontinued therapy due to toxicity; 6 neurotoxicity, 3 infection, 2 dermatological toxicity, 2 gastro-intestinal toxicity and 5 other toxicity. Discontinuation was comparable in patients ≥76 versus age ≤75, however none of the 10 patients > 80 years reached maintenance therapy, of whom 6/10 due to toxicity. Early mortality was higher in patients ≥76; 4/45 (9%) versus 1/75 (1%) in patients ≤75, mainly due to infections (4/5). The discontinuation rate due to toxicity was 1/25 (4%), 5/34 (15%) and 9/56 (16%)(p=0.34) in fit, unfit and frail patients respectively.ConclusionInduction treatment with ITd in NDMM results in high overall response rate of 81%, with 44% ≥ VGPR. Median time to response was 1.1 month. In frail and high risk cytogenetic patients induction therapy with ITd was equally effective with respect to response. Therapy was found to be feasible with limited toxicity and a low resulting discontinuation rate of 15%, although in patients >80 years toxicity was higher. [Display omitted] DisclosuresZweegman:Janssen: Other: advisory board participation, Research Funding; Celgene: Other: advisory board participation, Research Funding; Amgen: Other: advisory board participation; Takeda: Other: advisory board participation, Research Funding. Waage:Celgene, Takeda: Honoraria. Sonneveld:Celgene, Amgen, Janssen, Karyopharm, Takeda: Consultancy, Honoraria, Research Funding; Celgene Corporation, Amgen, Janssen, Karyopharm, SkylineDx, PharmaMar: Consultancy; Celgene Corporation, Amgen, Janssen, Karyopharm, PharmaMar, SkylineDx: Honoraria. Abildgaard:Takeda: Research Funding.

The Oral Proteasome Inhibitor Ixazomib, Alone and in Combination with Ibrutinib, Induces Lethality in Waldenstrom Macroglobulinemia Cells That Are Resistant to Ibrutinib

Introduction: Dysregulated B-cell receptor (BCR) signaling drives proliferation of Waldenstrom macroglobulinemia (WM) cells, which characteristically secrete excessive IgM. To maintain IgM production and preserve cellular homeostasis, WM cells rely on optimally functioning proteasomes to degrade and recycle proteins. This biological dependence has been clinically exploited using the proteasome inhibitor (PI), bortezomib (Btz), which is capable of inducing remission in 85% of WM patients (Treon et al Clin Canc Res, 2007) as a single agent. Despite, its activity, Btz use is associated with a high incidence of sensory and autonomic neuropathies as well as its cumbersome dosing schedule and non-oral route of administration. Thus, new agents that 1.) build on the activity of Btz, 2.) have a more favorable toxicity profile and 3.) can be combined with standard of care agents such as, ibrutinib (Ibr), are highly attractive and should be prioritized for investigation. Here we describe the preclinical activity of ixazomib (Ixa), a PI that is administered orally and is associated with significantly less peripheral neuropathy; in combination with Ibr, in WM cells.Methods: WM cell lines (BCWM.1 and RPCI.WM1) and their drug-resistant subclones were used. Genomic data including whole exome sequencing (WES) and copy number variations (CNV) was used in creation of digital simulation avatars of RPCI-WM1 and RPCI-WM1/IR (Ibr-resistant, Ibr-R variant), where the salient and prominently dysregulated cellular pathways were identified. Ixa +/- Ibr was tested in digital avatars and dose-dependent impact on simulated disease growth was assessed (Fig 1A). The predictive results were experimentally validated. Cell viability was measured using trypan blue or CellTiter Glo. Apoptosis was examined by annexin-V/PI staining of drug-treated cells followed by flow cytometry analysis. Mitochondrial membrane permeability (MOMP) was determined by TMRM staining of drug-treated cells followed by flow cytometry.Results: We first profiled the growth inhibitory action of Ixa in WM cell lines (n=9); all of which were sensitive to Ixa irrespective of acquired resistance to Btz, Ibr or carfilzomib with a median IC50 of 95.59nM. We then tested whether Ixa could cooperate with Ibr to enhance WM cell kill. Indeed, the Cellworks modeling tool, predicted a significant loss of viability and induction of apoptosis in both wildtype and Ibr-R cells treated with Ibr + Ixa, as compared to either single agent (Fig 1B-D). We confirmed this in vitro, where viability of wildtype WM cells treated with Ixa (20nM), Ibr (5uM) or Ibr + Ixa for 6h significantly decreased; most notable with Ixa + Ibr (25.5% viable cells). And this effect was similar in Ibr-R cell lines (30.3% viable cells) after combination treatment. Further confirming in silico predictive modeling results, we noted a significant increase in annexin-V/PI positivity in Ixa treated wildtype (24.5%) and Ibr-R WM cell lines (33.1%). Whereas Ibr alone exhibited minimal apoptosis in WM cell lines (~11 - 15%), the combination of with Ixa + Ibr enhanced programmed cell death in wildtype (40.2%) and Ibr-R cells (50.5%). As intrinsic apoptosis is triggered through mitochondrial disruption, we also examined mitochondrial transmembrane permeability (MOMP) in cells treated with Ibr, Ixa or Ixa + Ibr. In wildtype cells, mean MOMP induced by single agent Ixa was 23.5% and in Ibr-R cells 33%; increasing to 39% and 50.4%, respectively, after treatment with Ibr + Ixa. Mechanistic studies revealed changes in proteins regulated by the ubiquitin proteasome system and those associated with BTK signaling. While Ixa alone downregulated Bcl-2 and Mcl-1 levels, this effect was more marked in cells (both wildtype and Ibr-resistant) treated with Ixa + Ibr combination. Similar effects were noted in pPLCg2, albeit with some variability between the cell lines.Conclusion: Using a combination of in silico modeling and experimental approaches, we provide first preclinical evidence on the antitumor effects of Ibr combined with the oral PI, Ixa, in WM cells, including those resistant to Ibr. Whereas further investigation on the precise molecular mechanisms of how Ibr + Ixa cooperate to induce apoptosis in WM cell is ongoing, our studies provide the basis for clinical testing of the Ibr + Ixa regimen; with a phase II clinical study in development. [Display omitted] DisclosuresSingh:Cellworks Research India Pvt. Ltd: Employment. Kumar:Cellworks: Employment. Basu:Cellworks: Employment. Trivedi:Cellworks: Employment. Majumdar:Cellworks: Employment. Abbasi:Cellworks Group Inc.: Employment. Vali:Cellworks Group Inc.: Employment. Martin:Janssen: Consultancy, Honoraria, Other: travel expenses; Genentech: Consultancy; Celgene: Consultancy; Teva: Research Funding; Novartis: Consultancy; Gilead: Consultancy, Other: travel expenses. Ansell:Bristol-Myers Squibb: Research Funding; Merck: Research Funding; Celldex: Research Funding; Affimed: Research Funding; Seattle Genetics: Research Funding. Ailawadhi:Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Pharmacyclics: Research Funding; Takeda: Consultancy, Honoraria.

The Effect of the Proteasome Inhibitor Ixazomib (MLN9708) and UPR Mediated Therapy in Multiple Myeloma Cells and Bone Marrow Niche

Background: The concept of disrupting proteostasis in order to weaken cancer cells and challenge their microenvironment has shown striking clinical success, especially by applying proteasome inhibitors (PIs) in multiple myeloma (MM). The proteasome inhibitor MLN9708 (ixazomib) demonstrated significant preclinical and clinical anti-myeloma activity combined with a favorable toxicity profile. Proteostasis is also maintained by different adaptive pathways, including heat shock response (HSR) and the unfolded protein response (UPR). MM cells are characterized by mis- and overproduction and thus accumulation of immunoglobulin (Ig) within the bone marrow, relying on the UPR system to overcome stress due to this accumulation of unfolded proteins. The UPR mediates its function through three pathways, namely IRE-1α, PERK and ATF6. The most conserved UPR pathway is mediated by IRE-1α which reduces the ER stress load by splicing the XBP1 mRNA through removal of a 26-nucleotide intron.Aim: The UPR represents a vulnerability of MM cells which we aimed to harness with a novel combination therapy of UPR inhibition and proteasome inhibition.Methods: We used two MM cell lines (KMS11 and RPMI-8226) to validate the cytotoxic effects of ixazomib together with the IRE-1α inhibitors A106 and STF-083010 by measuring cell viability and proliferation, assessing synergistic or additive effects with the Bliss formula and quantifying the apoptotic cell fraction. For gene expression assays, total RNA was isolated and relative quantification of target gene expression was calculated by the comparative threshold cycle method.Results: Our in vitro results demonstrate that the combination of ixazomib and IRE-1α inhibitors has a synergistic effect on reducing of viability and proliferation of multiple myeloma cells and induces apoptosis as a single agent and in combination with IRE-1α inhibitors (Viability of KMS11 with ixazomib treatment 13.5% ± 0.35 vs 7.3% ± 0.23 combination with STF-083010, RPMI-8226 with ixazomib treatment 33.4% ± 0.56 vs 15.7% ± 0.07 combination with STF-083010; apoptotic fraction of KMS11 with ixazomib treatment 67.1% vs 76.9% combination with STF-083010, RPMI-8226 with ixazomib treatment 31.7% vs 36.3% combination with STF-083010). Ixazomib in combination with STF-083010 increased proteolytic cleavage of PARP, a signature of apoptosis and downstream target of Caspase-3, increased G1 phase regulators (p21cip1, p27kip1) and up-regulated pro-apoptotic regulator protein, BAX. In addition, we found that combination treatment increased the expression of pro-apoptotic BH3-only family members, PUMA and NOXA and decreased the expression of Bcl2 pro-survival family members (Bcl2, Bcl-xl).Mesenchymal stromal cells (MSCs) mimic the positive effects of bone marrow niche on MM cells in vitro . Co-culture of MM cells with Telomerase-immortalized Human Mesenchymal Stem Cell (hMSC-TERT) resulted in support of survival and cell growth of the two MM cell lines, (KMS11-hMSC-TERT with ixazomib treatment 55.4% ± 2.3 vs 24.2% ± 1.0 combination with STF-083010, RPMI-8226 with ixazomib treatment 68.6% ± 0.14 vs 17.8% ± 0.42 combination with STF-083010). However, the combination therapy of ixazomib with IRE-1α inhibitors significantly reduced the viability of multiple myeloma cells even when protected by the bone marrow niche microenvironment, confirming the notion that this combination therapy abrogates microenvironmental support of myeloma cells.Quantitative PCR (qRT-PCR) revealed a relevant up-regulation of XBP1s mRNA levels by ixazomib monotherapy in the two MM cell lines whereas the combination of proteasome inhibition by ixazomib with IRE-1α inhibition significantly reduced the expression level of XBP1s (KMS11 with ixazomib 77.3% ± 3.4 vs 34.8% ± 2.7 combination with STF-083010, RPMI-8226 with ixazomib 22.2.0% ± 1.7 vs 11.9% ± 1.7 combination with STF-083010), again demonstrating the IRE-1α pathway of the UPR as an important target in the therapy of multiple myeloma. A second experimental set with the IRE-1α inhibitor A106 showed similar results, for which data are not explicitly mentioned here.Conclusion: Taken together, our findings demonstrate that targeting the UPR specifically via inhibition of IRE-1α in combination with ixazomib is a promising therapeutic strategy in multiple myeloma. DisclosuresSalimi:Takeda: Consultancy, Research Funding. Schroeder:Takeda: Consultancy, Research Funding. Vieri:Takeda: Consultancy, Research Funding. Brümmendorf:Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Takeda: Consultancy, Research Funding. Appelmann:Takeda: Consultancy, Research Funding. Kharabi:Takeda: Consultancy, Research Funding.

Tourmaline US-MM6, an Open-Label, Single-Arm, Multicenter Study Evaluating the Effectiveness and Safety of Ixazomib in Combination with Lenalidomide and Dexamethasone (IRd) in Patients (pts) with Newly Diagnosed Multiple Myeloma (NDMM) Switching from a Bortezomib-Based Triplet Induction Regimen

Tourmaline US-MM6, an Open-Label, Single-Arm, Multicenter Study Evaluating the Effectiveness and Safety of Ixazomib in Combination with Lenalidomide and Dexamethasone (IRd) in Patients (pts) with Newly Diagnosed Multiple Myeloma (NDMM) Switching from a Bortezomib-Based Triplet Induction Regimen

Oral Proteasome Inhibitor Ixazomib for Switch-Maintenance Prophylaxis of Recurrent or Late Acute and Chronic Graft-versus-Host Disease after Day 100 in Allogeneic Stem Cell Transplantation

Graft-versus-host disease (GVHD) is a frequent complication in the first year after allogeneic stem cell transplantation (allo-HCT). Recipients of reduced-intensity (RI) or nonmyeloablative (NMA) conditioning combined with calcineurin inhibitor (CNI)-based GVHD prophylaxis frequently develop GVHD in the context of immunosuppression taper. Ixazomib is an oral proteasome inhibitor with a wide safety profile that has demonstrated immunomodulatory properties, inhibition of pro-inflammatory cytokines, and anti-tumor activity. We hypothesized that switch-maintenance GVHD prophylaxis using ixazomib would facilitate CNI taper without increased GVHD frequency and severity while maintaining graft-versus-tumor (GVT) effect and an acceptable safety profile. We conducted an open-label, prospective, single-center pilot study in patients with hematologic malignancies who received an RI or NMA conditioning and CNI-based GVHD prophylaxis that were within day 100 to 150 after HCT (n=18). Patients were treated with ixazomib once weekly on a 28-day cycle (3 weeks on, 1 week off). Treatment was safe; most adverse events were grade 1 or 2, with cytopenia and elevation in transaminases the most common. Five patients were removed from the study because of toxicity or side effects. Only 5 of 18 patients developed GVHD during the study, and its severity was driven by acute manifestations while chronic involvement was mild. The cumulative incidence of grade II-IV acute and chronic GVHD at 1-year after HCT was 33% (95% confidence interval [CI], 13-55). No patients died during the study, and only 1 had malignant relapse. An additional patient relapsed after completion of the study but within 1 year after HCT. The probability of progression-free survival and GVHD-free/relapse-free survival (composite endpoint) at 1 year were 89% (95% CI, 75-100) and 78% (95% CI, 61-100), respectively. Immune reconstitution analysis showed a rapid and sustained recovery in T-cell subpopulations and B cell reconstitution, and vaccine response in a subset of patients demonstrated continuing or de novo positive protective antibody titers. This study demonstrated low incidence of recurrent and late acute and chronic GVHD within 1 year after HCT possible associated with switch-maintenance GVHD prophylaxis using ixazomib. This approach allowed for CNI taper while preserving GVT effect, without aggravating GVHD. Our findings support further development of this approach and provide a proof-of-concept for switch-maintenance GVHD prophylaxis.

Oral ixazomib, lenalidomide, and dexamethasone for transplant-ineligible patients with newly diagnosed multiple myeloma

AbstractContinuous lenalidomide-dexamethasone (Rd)-based regimens are among the standards of care in transplant-ineligible newly diagnosed multiple myeloma (NDMM) patients. The oral proteasome inhibitor ixazomib is suitable for continuous dosing, with predictable, manageable toxicities. In the double-blind, placebo-controlled TOURMALINE-MM2 trial, transplant-ineligible NDMM patients were randomized to ixazomib 4 mg (n = 351) or placebo (n = 354) plus Rd. After 18 cycles, dexamethasone was discontinued and treatment was continued using reduced-dose ixazomib (3 mg) and lenalidomide (10 mg) until progression/toxicity. The primary endpoint was progression-free survival (PFS). Median PFS was 35.3 vs 21.8 months with ixazomib-Rd vs placebo-Rd, respectively (hazard ratio [HR], 0.830; 95% confidence interval, 0.676-1.018; P = .073; median follow-up, 53.3 and 55.8 months). Complete (26% vs 14%; odds ratio [OR], 2.10; P < .001) and ≥ very good partial response (63% vs 48%; OR, 1.87; P < .001) rates were higher with ixazomib-Rd vs placebo-Rd. In a prespecified high-risk cytogenetics subgroup, median PFS was 23.8 vs 18.0 months (HR, 0.690; P = .019). Overall, treatment-emergent adverse events (TEAEs) were mostly grade 1/2. With ixazomib-Rd vs placebo-Rd, 88% vs 81% of patients experienced grade ≥3 TEAEs, 66% vs 62% serious TEAEs, and 35% vs 27% TEAEs resulting in regimen discontinuation; 8% vs 6% died on study. Addition of ixazomib to Rd was tolerable with no new safety signals and led to a clinically meaningful PFS benefit of 13.5 months. Ixazomib-Rd is a feasible option for certain patients who can benefit from an all-oral triplet combination. This trial was registered at www.clinicaltrials.gov as #NCT01850524.

Pre-Clinical Evaluation of the Proteasome Inhibitor Ixazomib against Bortezomib-Resistant Leukemia Cells and Primary Acute Leukemia Cells

At present, 20–30% of children with acute leukemia still relapse from current chemotherapy protocols, underscoring the unmet need for new treatment options, such as proteasome inhibition. Ixazomib (IXA) is an orally available proteasome inhibitor, with an improved safety profile compared to Bortezomib (BTZ). The mechanism of action (proteasome subunit inhibition, apoptosis induction) and growth inhibitory potential of IXA vs. BTZ were tested in vitro in human (BTZ-resistant) leukemia cell lines. Ex vivo activity of IXA vs. BTZ was analyzed in 15 acute lymphoblastic leukemia (ALL) and 9 acute myeloid leukemia (AML) primary pediatric patient samples. BTZ demonstrated more potent inhibitory effects on constitutive β5 and immunoproteasome β5i proteasome subunit activity; however, IXA more potently inhibited β1i subunit than BTZ (70% vs. 29% at 2.5 nM). In ALL/AML cell lines, IXA conveyed 50% growth inhibition at low nanomolar concentrations, but was ~10-fold less potent than BTZ. BTZ-resistant cells (150–160 fold) displayed similar (100-fold) cross-resistance to IXA. Finally, IXA and BTZ exhibited anti-leukemic effects for primary ex vivo ALL and AML cells; mean LC50 (nM) for IXA: 24 ± 11 and 30 ± 8, respectively, and mean LC50 for BTZ: 4.5 ± 1 and 11 ± 4, respectively. IXA has overlapping mechanisms of action with BTZ and showed anti-leukemic activity in primary leukemic cells, encouraging further pre-clinical in vivo evaluation.

Abstract PO039: Combined histone deacetylase and proteasome inhibition in patient-derived endometrial cancer organoid models promotes massive cell death

Abstract Patient-derived organoids (PDOs) are a promising model for personalized cancer treatment. Accumulating evidence indicates that PDOs can predict clinical outcomes in patients. A handful studies in several cancer types have established that PDOs can recapitulate both histological and genomic features of the lesion from which they were derived. Additionally, PDOs can grow with high efficiency in a short time, enabling prediction of responsiveness to chemotherapy and development of more targeted therapies for patients. Our long-term goal is to create a PDO biorepository comprising all major histopathological subtypes of endometrial cancer, perform drug screening on these PDOs, and identify novel single agent and combinatorial regimens to advance into clinical trials. Herein we describe progress to date on generation of endometrial PDOs and drug screening to identify potential therapeutic options. First, we created PDO cultures from seven patients with different histological subtypes of endometrial cancer, including early stage/grade endometrioid adenocarcinoma and high-grade serous carcinoma. Next, we exposed each PDO to a panel of 23 chemotherapeutic agents and targeted drugs. Surprisingly, despite a divergent response to standard chemotherapy (platinum and taxane compounds), all PDOs showed high responsiveness to the combination of a histone deacetylase (HDAC) inhibitor and a proteasome inhibitor. Specifically, we achieved nearly complete cell killing within 72 hrs of exposure of organoid cultures to HDAC and proteasome inhibitors, with concentrations in the low nanomolar range. Similar results were achieved with different proteasome inhibitors (bortezomib and ixazomib) and HDAC inhibitors (romidepsin and belinostat). At present, these classes of agents have primarily been studied in multiple myeloma, though multiple clinical trials of combined proteasome and HDAC inhibitor therapy are active in advanced solid tumors and lymphomas. HDAC inhibitors maintain the acetylation of histones and thereby increase the expression of genes associated with apoptosis and cell cycle arrest. Proteasome inhibitors prevent proteasomal degradation, leading to upregulation of proapoptotic protein expression, cell cycle arrest, and ER stress, downregulation of angiogenesis and the pro-inflammatory protein NF-κB, and ROS generation. Mechanistically, HDAC inhibitors and proteasome inhibitors promote dual proteasome and aggresome blockage and induce apoptosis due to the accumulation of misfolded proteins. Our findings of profound tumor cell death in multiple different PDOs serve as the foundation for a future clinical trial using the HDAC inhibitor ixazomib and proteasome inhibitor romidepsin in advanced and recurrent endometrial cancers as well as high-grade serous ovarian cancer, which shares histologic and molecular features. Concomitantly, we are investigating the in vitro and in vivo effects of the combination on the induction of apoptosis and endometrial tumor regression. Citation Format: Jianling Bi, Kristina W. Thiel, Eric J. Devor, Andreea M. Newtson, Yuping Zhang, Kimberly K. Leslie. Combined histone deacetylase and proteasome inhibition in patient-derived endometrial cancer organoid models promotes massive cell death [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO039.