Second-generation Proteasome Inhibitor Research Articles

Practical Aspects of the Use of Carfilzomib in Multiple Myeloma

Carfilzomib (Kyprolis®, Amgen), a second-generation proteasome inhibitor, is capable of covalent bonding and irreversible inhibition of the 20S proteasome chymotrypsinlike activity. In 2016 this drug was approved in Russia for monotherapy of relapsed refractory multiple myeloma (MM) and in combination with lenalidomide and dexamethasone (KRd) or only with dexamethasone (Kd) for treatment of patients with relapsed MM after at least one line of prior therapy. The present review outlines mechanism, clinical efficacy, and adverse effects of carfilzomib according to the data of a phase II (monotherapy) trial and two key randomized phase III (carfilzomib combined with other drugs) trials. The ASPIRE trial demonstrated that adding carfilzomib to the combination of lenalidomide and dexamethasone (KRd) significantly improves progression-free survival (PFS) compared with the Rd original regimen (median 26.3 vs. 17.6 months; hazard ratio [HR] 0.69; p = 0.0001). Median overall survival (OS) was 48.3 months (95% confidence interval [95% CI] 42.4-52.8 months) for KRd vs. 40.4 months (95% CI 33.6-44.4 months) for Rd (HR 0.79; p = 0.0045). The ENDEAVOR trial showed that as compared with combination of bortezomib and dexamethasone (Vd) the carfilzomib + dexamethasone (Kd) regimen significantly improves PFS (median 18.7 vs. 9.4 months; HR 0.53; p < 0.0001) and OS (47.6 vs. 40.0 months; HR 0.79; p = 0.010) as well. The present review also discusses how carfilzomib is to be used in special patient groups (with renal failure and high cytogenetic risk).

Encouraging Outcomes for Patients with Multiple Myeloma Treated in Early Phase Clinical Trials: A Single Centre and Meta-Analysis

Introduction:The overall survival (OS) for patients with multiple myeloma (MM) continues to improve with advances in therapies such as second-generation proteasome inhibitors (PI), immunomodulatory agents (IMiDs) and monoclonal antibodies. Advanced therapeutics (eg chimeric antigen receptor (CAR-T) cells), immunotherapies and 3rd generation agents are also under evaluation. Therefore, interest in Phase 1 clinical trials has increased as patients may benefit from exposure to a novel therapy. Also, increasingly data has been used to obtain breakthrough designation from regulatory authorities as part of accelerated approvals. However, these therapies have unproven efficacy, may cause potentially severe toxicities and patients may receive sub-optimal doses. We therefore analysed the outcomes of our patients and to complement this performed a meta-analysis of publicly available Phase I MM trials.Methods: This was a retrospective review of patients with MM enrolled onto phase I and I/II trials at the NIHR/UCLH Clinical Research Facility, London, UK. Patients received at least one dose of study drug, AEs were graded by CTCAE 4.0 and response criteria evaluated as per protocol. Individual-patient survival data were analysed using the Kaplan-Meier method and survival curves compared with the log-rank test. A meta-analysis using Medline, EMBASE and clinicaltrials.gov was performed to identify abstracts/papers reporting phase I trials from January 2016 to December 2017. Data was extracted by two independent reviewers using the same inclusion and exclusion criteria with significant overlap to ensure inter-observer consistency. Meta-analyses of Overall Response Rate (ORR) were performed using random-effect models.Results: 50 patients were enrolled onto 7 phase I or I/II clinical trials between March 2012 to June 2018 at the NIHR/UCLH Clinical Research Facility. 6 participated in 2 trials (each trial entry considered a separate event). IMPs were: monoclonal antibody (Mab) (1), HDAC inhibitor (1), immune-modulator (1), proteasome pathway inhibitors (2), kinase pathway inhibitor (1), epigenetic modifier (1). 3 trials were monotherapy and 4 as combinations. The median age was 54 years (33-73), 46% were female. The median time from diagnosis to trial entry was 58.5m (IQR 36-104.5) and median prior lines of therapy was 3 (0-7). Patients received a median of 10 cycles (1-34) over a median duration of 176 days (IQR 112-425). Study withdrawal was due to progressive disease (50%), autograft (16.1%), toxicity (12.5%), completion of study (12.5%) and other (3.6%) (Table 1).30.4% of patients experienced G3-4 toxicity and 28.6% had dose interruptions of ≥7 days. The overall response rate (≥PR) was 55.3%, ≥VGPR: 26.7% (see Table 1).The median PFS was 14.2 m (95% CI:7.6-15.9) and OS 37.3m (95% CI:29.2-not reached) with a median follow-up of 26.5m. Median OS for those with < 4 prior lines was better (48.1m) vs those ≥ 4 prior lines (30.6m, p-value log-rank test=0.03). Those with adverse risk cytogenetics appeared to do worse vs standard risk: PFS 14.2 vs 7.6m (log rank not significant); OS 37.3 vs 30.6m (log rank: not significant). Most patients (80.3%) received further treatment after trial (12.5% received another trial, 46.4% chemotherapy; 21.4%: autograft). 8.9% were managed expectantly and 5.4% palliated.110 trial abstracts & publications were selected for meta-analysis that included 3222 patients with a median age of 64 years (30-75), 42% female, median 4 prior lines. 64.6% of reports were combination studies. 17% targeted the proteasome, 17% were MAbs, 10.9% were epigenetic modifiers, 5.5% were cellular therapies and 24.6% were other small molecule inhibitors (Table 2).The overall response rate across all studies was 52.2% (95% CI 46-58.2). Higher responses were described for immunotherapy studies: BCMA CAR-T cells, 74.2% (95%CI 42.3- 91.8) and monoclonal antibodies, 64.2% (95% CI 56.2- 71.4). PFS and OS was not consistently reported across studies.Conclusions: Patients derive benefit from phase 1 trials with clinically significant responses reported from this single centre and meta-analysis. Additionally, most received further treatment after. The best responses were reported for immunotherapy studies. Despite this, in our data there was an overall trend for worse outcomes for adverse risk cytogenetics and for those treated later in their disease course. DisclosuresTownsend:Gilead: Consultancy, Honoraria; Roche: Consultancy, Honoraria. Yong:Takeda: Speakers Bureau; Amgen: Honoraria, Research Funding, Speakers Bureau; Celgene: Honoraria; Janssen: Honoraria, Speakers Bureau. Popat:Amgen: Honoraria.

Pre-Clinical Trial to Evaluate the Efficacy of Delayed Administration of Ixazomib in the Prophylaxis of Chronic Graft-Versus-Host Disease

Introduction: Although survival rates have improved over the years, chronic graft-versus-host disease (cGvHD) remains the most frequent and severe complication in the long term after allogeneic hematopoietic stem cell transplantation (allo-HSCT). All the strategies developed to reduce its incidence are based on procedures aimed to decrease the risk of acute GvHD that, consequently, can also reduce the risk of cGvHD, mainly using immunosuppression in the early post-transplant period. These strategies induce apoptosis of donor T lymphocytes, responsible for a/cGvHD but also for graft-versus-tumor response. In the present project, we have evaluated a new strategy aimed to reduce the risk of cGvHD by manipulating the immune response not in the early post-transplant period, but in later phases.cGvHD develops via a complex cellular and molecular network involving thymus damage and unusual antigen presentation leading to aberrant T- and B-cell reactions characterized by Th17/Tc17 differentiation, macrophage sequestration in tissue, alloantibody formation, and fibrosis. Most of these cell populations are dependent on NF-kB for their activation. Ixazomib is a second-generation proteasome inhibitor for oral administration, representing an ideal candidate for prophylaxis in GvHD.Objective: We propose to develop a murine model of cGVHD and progressive onset cGvHD to test the efficacy of delayed administration of ixazomib as a novel strategy to decrease the risk of cGvHD.Methods: For in vitro studies, peripheral blood mononuclear cells from healthy donors were stimulated in the presence of different concentrations of ixazomib. After 48h and 120h, apoptosis was analyzed, and activation markers were evaluated.For in vivo studies, a murine model of progressive cGvHD (pcGvHD - allowing the recipient to survive to mild aGvHD, thus favoring autoreactive T cells to expand and cause cGvHD) and a scleroderma model of cGvHD were used. Ixazomib at 0.75mg/Kg/twice weekly, 2X from day +21 in the pcGvHD, with or without cyclosporine A (CyA) at 5mg/Kg/day from day 0 until the end of the study. For scleroderma cGvHD group, 3mg/Kg/2X from day +30 was used up to 120 days post-transplant. Flow cytometry was used to evaluate the different lymphocyte populations in the different target organs of the GvHD.Results:In vitro results showed that activated T lymphocytes are sensitive to the proapoptotic effect of ixazomib (>100nM), while high concentrations of the drug are necessary to cause apoptosis in the non-activated cells (5000nM). Through CD27 and CD45 expression, we verified that ixazomib has a proapoptotic effect mainly on naïve and effector cells. We also verified a decrease in the activation markers of CD3+CD25+INF-γ+ cells (p˂0.01, 1000nM, n = 4).In the animal trial, the survival of the pcGvHD model mice treated with ixazomib was significantly higher as compared to untreated mice, with a significant decrease in the signs of pcGvHD (Figure 1A). In addition, the combination of CyA and ixazomib improved survival as compared to those mice receiving CyA or ixazomib alone as well as untreated controls (Figure 1B). In the scleroderma cGvHD model, we observed that the animals treated with ixazomib showed significantly less signs of GvHD (p˂0.0001) (Figure 2).Multiparametric flow cytometer analyses showed that in the pcGvHD model, mice treated with ixazomib had a decrease of effector CD4 T-cells in bone marrow (p=0.06) and spleen (p=0.015) when compared to untreated mice. Also an increase of CD19+ cells in the colon (p=0.04), liver (p=0.035), lymph nodes (LN) (p=0.037) and lungs (p=0.03) was observed. Regarding the regulatory T cells (Foxp3+), the treated mice had a significant increase in LN (p=0.02), Peyer patches (p=0.015) and thymus (p=0.028) as compared to untreated mice.Conclusion:In vitro studies show that ixazomib induces apoptosis on activated T lymphocytes and decreases the expression of activation markers. Our in vivo model indicates that the combination of CyA and ixazomib for the prevention of pcGvHD and cGVHD after allogeneic HSCT is promising and merits further investigation in clinical trials.Figure 1. Survival of pcGvHD.Figure 2. Score graphic of scleroderma cGvHD. BM - Bone marrow, mice that were transplanted with BM from BALBc mice (Syngeneic). [Display omitted] DisclosuresRamos:Takeda Oncology: Research Funding.

Carfilzomib-Based Regimens Are Efficacious and Well Tolerated in Heavily Pre-Treated Multiple Myeloma Patients: Real World Data from a Norwegian Cohort

IntroductionMultiple myeloma (MM) is considered an incurable disease. However, during the last years the survival has improved, partly due to the development of new drugs. The second-generation proteasome inhibitor Carfilzomib (Kyprolis®) was first approved for marketing in Norway in January 2016. Carfilzomib in combination with Dexamethasone (Kd) for patients with relapsing and/or refractory multiple myeloma (RRMM) was approved for reimbursement in August 2017, while the combination with Lenalidomide (Revlimid®) and Dexamethasone (KRd) is still not reimbursed in Norway. Both regimens now have mature and beneficial survival data (vs Vd and Rd, respectively). In the period before market approval, Carfilzomib was provided through a named patient program (NPP) for patients who had a critical disease with few treatments options. This abstract presents results from this cohort.AimIn this retrospective study all 33 Norwegian MM patients who received Carfilzomib through a named patient program (NPP) during the period of March 2014 to January 2016, were included. The aim was to describe the patients 'characteristics and their treatment responses, in addition to use the real-life experience to highlight a possible benefit of making new drugs available before marketing approval. During this study period, neither Daratumumab, Ixazomib nor Elotuzumab were available in Norway. The medical records were printed and sent from 10 different hospitals to Oslo Myeloma Center. Baseline values and treatment responses was assessed and entered into our myeloma database. Statistical analysis was done to determine response rates and time to event outcomes.ResultsThe participants had received a median of 6 (1-11) previous lines of therapy, including bortezomib (100%), and thalidomide and/or lenalidomide (97%) before inclusion in this study Among these, 64 % or 66 % respectively was refractory to the indicated treatment. 76 % of the study participants had received at least one ASCT. Median time from diagnosis to initiating carfilzomib treatment was 5 years (0-15) for the 32 patients who received carfilzomib. Patients who received the triplet treatment KRd, had a benefit in terms of response with ORR of 19%, compared to 8 %for patients receiving Kd/K (p=0, 04). CBR for these patients was 22 % and 25 %, respectively (p=0,33). There was no difference between the two groups for PFS or OS, with a median OS of 10 months (0-24) for patients receiving KRd compared to 9 month (2-14) for Kd/K. However, patients receiving KRd showed a trend towards a better PFS compared to Kd with a median PFS of 3,5 months (1,4 - 4,6) compared to 2,5 months (0,2 - 3,8) (p=0,095).Adverse events occurred in 91 % of the patients where 41 %, 31 % and 41 % respectively experienced a Grade 3 or higher for anemia, neutropenia or thrombocytopenia (according to CTCAE vs 4.0). 19 % experienced cardiovascular complications and 3% ended treatment due to them. Of these patients, 50 % had received doxorubicin at a previous treatment line and 17% received doxorubicin in combination with Carfilzomib. Overall, 9% of the patients discontinued the treatment due to adverse events while 6 % died before the end of the treatment. Carfilzomib was not considered a direct cause of death in any of these patients. At the end of the study (January 2018), 76 % of the participants were dead.ConclusionIn this real-life study, RRMM patients who received lenalidomide in addition to carfilzomib and dexametason showed a superior response in comparison with patients who received carfilzomib and dexamethasone with an ORR of 19 % versus 8 %. There was no significant overall survival benefit, but a trend towards a better PFS for KRd compared to Kd/K. As an overall conclusion, this group of highly treated MM patients had a benefit from receiving Carfilzomib in combination with lenalidomide before marketing approval. DisclosuresSchjesvold:Oncopeptides: Consultancy; Novartis: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Adaptive: Consultancy; Bayer: Consultancy; Bristol Myers Squibb: Consultancy; Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria.

Carfilzomib-Lenalidomide-Dexamethasone (KRd) Induction-Autologous Transplant (ASCT)-Krd Consolidation Vs KRd 12 Cycles Vs Carfilzomib-Cyclophosphamide-Dexamethasone (KCd) Induction-ASCT-KCd Consolidation: Analysis of the Randomized Forte Trial in Newly Diagnosed Multiple Myeloma (NDMM)

Background: Proteasome inhibitor (PI)-based induction and consolidation proved to be effective in newly diagnosed multiple myeloma (NDMM) patients (pts) eligible for melphalan 200 mg/m2-autologous stem cell transplant (MEL200-ASCT). High response rates have been reported with the second-generation PI Carfilzomib in combination with Lenalidomide-dexamethasone (KRd) or Cyclophosphamide-dexamethasone (KCd). Aims: The primary aim was to evaluate the efficacy and safety of KRd induction-ASCT-KRd consolidation (KRd-ASCT-KRd) vs 12 cycles of KRd (KRd12) vs KCd induction-ASCT-KCd consolidation (KCd-ASCT-KCd). Methods: NDMM pts ≤65 years were randomized (1:1:1; stratification ISS and age) to: KRd-ASCT-KRd: 4 28-day cycles with KRd induction (Carfilzomib 20/36 mg/m2 IV days 1,2,8,9,15,16; Lenalidomide 25 mg days 1-21; dexamethasone 20 mg days 1,2,8,9,15,16) followed by MEL200-ASCT and 4 KRd consolidation cycles; KRd12: 12 KRd cycles; KCd-ASCT-KCd: 4 28-day induction cycles with KCd (Carfilzomib 20/36 mg/m2 IV days 1,2,8,9,15,16; Cyclophosphamide 300 mg/m2 days 1,8,15; dexamethasone 20 mg days 1,2,8,9,15,16) followed by MEL200-ASCT and 4 KCd consolidation cycles. Thereafter, pts were randomized to maintenance with Lenalidomide alone or plus Carfilzomib. Centralized minimal residual disease (MRD) evaluation - 8-color second generation flow cytometry, sensitivity 10-5 - was performed in pts achieving ≥very good partial response (VGPR). Endpoints were pre-maintenance stringent complete response (sCR) and MRD negativity in intention-to-treat (ITT) analysis. Data cut-off was May 30, 2018. Results: 474 NDMM pts were randomized (KRd-ASCT-KRd, n=158; KRd12, n=157; KCd-ASCT-KCd, n=159) and analyzed. Pts characteristics were well balanced. Median follow-up was 20 months. Depth of response improved during treatment (Figure). By ITT analysis, rates of pre-maintenance sCR was similar between KRd-ASCT-KRd (41%) and KRd12 (42%), and significantly higher than with KCd-ASCT-KCd (30%; P value KRd-ASCT-KRd vs KCd-ASCT-KCd=0.047; P value KRd12 vs KCd-ASCT-KCd=0.028). Similarly, rate of ≥CR was 49% with KRd-ASCT-KRd, 52% with KRd12 and 38% with KCd-ASCT-KCd (P value KRd-ASCT-KRd vs KCd-ASCT-KCd=0.041; P value KRd12 vs KCd-ASCT-KCd=0.014) and rate of ≥CR+unconfirmed CR (missing immunofixation confirmation) raised to 60% vs 63% vs 46% in the 3 groups, respectively; rate of ≥VGPR was 88% with KRd-ASCT-KRd, 86% with KRd12 and 74% with KCd-ASCT-KCd (P value KRd-ASCT-KRd vs KCd-ASCT-KCd=0.002; P value KRd12 vs KCd-ASCT-KCd=0.008). In multivariate analysis, the main factor affecting probability of achieving ≥VGPR, ≥CR or sCR was treatment with KRd-ASCT-KRd or KRd12 vs KCd, with no significant impact of ISS Stage or FISH abnormalities. In ITT analysis (MRD missing [31/395 VGPR pts, 8%] and Conclusions: Rates of MRD negativity, sCR, ≥CR, ≥VGPR were significantly higher with KRd-ASCT-KRd and KRd12 vs KCd. At present, no differences in MRD and overall best response (sCR, ≥CR, ≥VGPR) were noticed between KRd-ASCT-KRd and KRd12; longer follow-up is needed to evaluate survival. Treatment was well tolerated. Updated data will be presented at the meeting. Disclosures Gay:Roche: Other: Advisory Board; Seattle Genetics: Other: Advisory Board; Bristol-Myers Squibb: Honoraria; Janssen: Honoraria; Celgene: Honoraria, Other: Advisory Board; Amgen: Honoraria; Takeda: Honoraria, Other: Advisory Board. Galli:Sigma-Tau: Honoraria; Janssen: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria. Belotti:Celgene: Other: Advisory Board; Amgen: Other: Advisory Board. Zamagni:BMS: Honoraria, Membership on an entity9s Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity9s Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity9s Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity9s Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity9s Board of Directors or advisory committees. Angelucci:Novartis: Honoraria, Other: Chair Steering Committee TELESTO protocol in MDS; Celgene: Honoraria, Other: Chair DMC proptocol BELIEVE 1 and BELIVE 2 in Thalassemia; Vertex Pharmaceuticals Incorporated (MA) and CRISPR Therapeutics AG (CH): Other: Chair DMC CRISPR CAS9 in Hemoglobinopathies; Jazz Pharmaceuticals Italy: Other: Local (national) advisory board on AML; Roche Italia: Other: Local (national) advisory board on biosimilars. Annibali:Celgene; Takeda; Amgen, Janssen Cilag: Honoraria. Offidani:Amgen: Honoraria, Other: Advisory Board; Takeda: Honoraria, Other: Advisory Board; Janssen: Honoraria, Other: Advisory Board; Celgene: Honoraria, Other: Advisory Board; Bristol-Myers Squibb: Honoraria, Other: Advisory Board. Palumbo:Takeda: Employment. Musto:Amgen: Honoraria; BMS: Honoraria; Takeda: Honoraria; Janssen: Honoraria; Celgene: Honoraria. Cavo:GlaxoSmithKline: Honoraria, Membership on an entity9s Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity9s Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity9s Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity9s Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity9s Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity9s Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Membership on an entity9s Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Honoraria, Membership on an entity9s Board of Directors or advisory committees. Boccadoro:Bristol-Myers Squibb: Honoraria, Research Funding; Mundipharma: Research Funding; Sanofi: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; AbbVie: Honoraria.

Ixazomib in combination with carboplatin in pretreated women with advanced triple-negative breast cancer, a phase I/II trial of the AGMT (AGMT MBC-10 trial)

BackgroundTriple-negative breast cancer (TNBC) comprises a heterogeneous group of diseases which are generally associated with poor prognosis. Up to now, no targeted treatment beyond anti-VEGF therapy has been approved for TNBC and cytotoxic agents remain the mainstay of treatment. Ixazomib is a selective and reversible inhibitor of the proteasome, which has been mainly investigated in the treatment of multiple myeloma. In a preclinical study TNBC cells were treated with the first-generation proteasome inhibitor bortezomib in combination with cisplatin and synergistic efficacy was demonstrated. Clinical data are available for carboplatin plus bortezomib in metastatic ovarian and lung cancers showing remarkable antitumor activity and good tolerability (Mol Cancer 11:26 2012, J Thorac Oncol 4:87–92 2009, J Thorac Oncol 7:1032–1040, 2012). Based on this evidence, the phase I/II MBC-10 trial will evaluate the toxicity profile and efficacy of the second-generation proteasome inhibitor ixazomib in combination with carboplatin in patients with advanced TNBC.MethodsPatients with metastatic TNBC pretreated with at least one prior line of chemotherapy for advanced disease with a confirmed disease progression and measurable disease according to RECIST criteria 1.1 are eligible for this study. Patients will receive ixazomib in combination with carboplatin on days 1, 8, and 15 in a 28-day cycle. The phase I part of this study utilizes an alternate dose escalation accelerated titration design. After establishing the maximum tolerated dose (MTD), the efficacy and safety of the combination will be further evaluated (phase II, including 41 evaluable patients). All patients will continue on study drugs until disease progression, unacceptable toxicity or discontinuation for any other reason. Primary endpoint of the phase II is overall response rate, secondary endpoints include progression-free survival, safety, and quality of life. This trial is open for patient enrollment since November 2016 in six Austrian cancer centers. Accrual is planned to be completed within 2 years.DiscussionBased on preclinical and clinical findings an ixazomib and carboplatin combination is thought to be effective in metastatic TNBC patients. The MBC-10 trial is accompanied by a broad biomarker program investigating predictive biomarkers for treatment response and potential resistance mechanisms to the investigational drug combination.Trial registrationEudraCT Number: 2016–001421-13 received on March 31, 2016, ClinicalTrials.gov Identifier: NCT02993094 first posted on December 15, 2016. This trial was registered prospectively.

Proteomic approach for understanding milder neurotoxicity of Carfilzomib against Bortezomib

The proteasomal system is responsible for the turnover of damaged proteins. Because of its important functions in oncogenesis, inhibiting the proteasomal system is a promising therapeutic approach for cancer treatment. Bortezomib (BTZ) is the first proteasome inhibitor approved by FDA for clinical applications. However neuropathic side effects are dose limiting for BTZ as many other chemotherapeutic agents. Therefore second-generation proteasome inhibitors have been developed including carfilzomib (CFZ). Aim of the present work was investigating the mechanisms of peripheral neuropathy triggered by the proteasome inhibitor BTZ and comparing the pathways affected by BTZ and CFZ, respectively. Neural stem cells, isolated from the cortex of E14 mouse embryos, were treated with BTZ and CFZ and mass spectrometry was used to compare the global protein pool of treated cells. BTZ was shown to cause more severe cytoskeletal damage, which is crucial in neural cell integrity. Excessive protein carbonylation and actin filament destabilization were also detected following BTZ treatment that was lower following CFZ treatment. Our data on cytoskeletal proteins, chaperone system, and protein oxidation may explain the milder neurotoxic effects of CFZ in clinical applications.

Carfilzomib-Lenalidomide-Dexamethasone in the Management of Lenalidomide-Refractory Multiple Myeloma

Carfilzomib-Lenalidomide-Dexamethasone in heavily pretreated Multiple Myeloma, refractory to lenalidomide.

Proteasome inhibitors for multiple myeloma

Therapeutic strategies for multiple myeloma have dramatically changed in the last two decades, especially after the introduction of proteasome inhibitors. The first-in-class proteasome inhibitor, bortezomib, was approved by the US Food and Drug Administration in 2003. Since then, it has been a backbone therapy for not only relapsed or refractory myeloma patients but also newly diagnosed multiple myeloma patients. Second-generation proteasome inhibitors, such as carfilzomib and ixazomib, have been approved, and three proteasome inhibitors were incorporated into several regimens with other cytotoxic agents, such as alkylating agents, immunomodulatory drugs and monoclonal antibodies. Because each proteasome inhibitor shows different properties with respect to adverse events, understanding and managing each adverse event of proteasome inhibitors are necessary for the continuation of therapy with minimal interruption of treatment. This review summarizes the recent advances in proteasome inhibitors used in the treatment of multiple myeloma.

PO-039 Examination of the anti-tumour and neuropathic side effect responses of first generation and second generation proteasome inhibitors

IntroductionThe overall goal in cancer treatment is to damage cancer cell macromolecules that causes the cancer cells to die. But in the treatment process, the defense mechanisms developed by the cancer cells affect the response to the treatment. As we have shown in our studies, cancer cells after anti-cancer stress have about 10–20 times more proteasome activation than healthy cells. Therefore, proteasome inhibition plays an important role in the prevention of resistance and recurrence in cancer treatment.Bortezomib, the first generation proteasome inhibitor, is used intensively, in hematologic cancer types, and significantly increases patient survival. However, it causes significant side effects such as neuropathy. New proteasome inhibitors are being produced to reduce side effects in the market. These inhibitors include clinical studies as well as preclinical studies. However, the lack of side-effect studies causes the early termination of clinical trials.Material and methodsIn this study, we compared the anti-tumour effects and neural toxicities of first generation and second-generation proteasome inhibitors in co-culture model. In this direction, human neural progenitor cells and K562 leukaemia cells were used. Co-cultures were incubated with 100 nM proteasome inhibitors for 24 hour and effects on the cancer cells and neural cells were analysed separately. Apoptotic cell death was evaluated with Annexin V/PI double staining by flow cytometry in K562 cells, and also PARP, Caspase 9 protein levels have been analysed in both cells. Protein oxidation related parameters such as protein carbonyls, ubiquitinated proteins, and HSP levels were investigated for understanding the stress response in both cells. Additionally, cytoskeleton proteins β-actin and β- tubulin changes were assessed in neural cells with confocal microscopy.Results and discussionsOur data showed that new proteasome inhibitors are less toxic in neural cells when compared to bortezomib. Protein carbonyls and ubiquitinated proteins were highest in bortezomib treated cells. On the other hand, especially carfilzomib has a lower anti-tumour activity in K562 cells when compared to bortezomib.ConclusionThis study may bring a highlight for the clinical usage and side effects of new proteasome inhibitors.

Abstract CT160: Ixazomib in combination with carboplatin in pretreated women with advanced triple negative breast cancer, an ongoing phase I/II trial (AGMT MBC-10 trial)

Abstract Background: Triple-negative breast cancer (TNBC) comprises a heterogeneous group of diseases which are generally associated with poor prognosis. Recently, the PARP inhibitor olaparib was approved as first targeted treatment beyond antiVEGF therapy for the BRCA1/2 mutated subgroup of TNBC. However, cytotoxic agents still remain the mainstay of treatment for this breast cancer subtype. Ixazomib is a selective and reversible inhibitor of the proteasome, which has been mainly investigated as treatment of multiple myeloma. In a preclinical cell line model for TNBC the first-generation proteasome inhibitor bortezomib showed synergistic efficacy with cisplatin. Clinical data are available for carboplatin plus bortezomib in metastatic ovarian and lung cancers showing remarkable antitumor activity (47% and 38% response rate, respectively) and good tolerability. In solid tumors cytotoxic effect of proteasome inhibitors is thought to be mediated through different mechanisms: (1) Inhibition of the Fanconi Anemia and BRCA1 DNA repair mechanism (2) Inhibition of p53 degradation (3) Inhibition of NF-kappa B signaling cascade. Based on this evidence, the phase I/II MBC-10 trial will evaluate the toxicity profile and efficacy of the oral second-generation proteasome inhibitor ixazomib in combination with carboplatin in patients with advanced TNBC. Trial Design: Patients with metastatic TNBC pretreated with at least one prior line of chemotherapy for advanced disease with a confirmed disease progression and measurable disease are eligible for this study. Patients will receive ixazomib in combination with carboplatin on days 1, 8, and 15 in a 28-day cycle. The phase I part of this study uses an alternate dose escalation accelerated titration design. After establishing the maximum tolerated dose (MTD), accrual continues to evaluate the efficacy and safety of the combination (phase II, including 41 evaluable patients). All patients will continue on study drugs until disease progression, unacceptable toxicity or discontinuation for any other reason. Primary endpoint of the phase II is overall response rate, secondary endpoints include safety profile, progression-free survival and quality of life. The MBC-10 trial is accompanied by a broad biomarker program investigating predictive biomarkers for treatment response and potential resistance mechanisms to the investigational drug combination. This trial is open for patient enrollment since November 2016 in six Austrian cancer centers. Accrual is planned to be completed within two years. ClinicalTrials.gov Identifier: NCT02993094 Citation Format: Gabriel Rinnerthaler, Simon P. Gampenrieder, Andreas Petzer, Renate Pusch, Daniela Voskova, Dieter Rossmann, Marija Balic, Daniel Egle, Holger Rumpold, Christian F. Singer, Thomas Melchardt, Hanno Ulmer, Brigitte Mlineritsch, Richard Greil. Ixazomib in combination with carboplatin in pretreated women with advanced triple negative breast cancer, an ongoing phase I/II trial (AGMT MBC-10 trial) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT160.

Efficacy and safety of carfilzomib in relapsed and/or refractory multiple myeloma: systematic review and meta-analysis of 14 trials.

ObjectiveCarfilzomib (Carf) is a second-generation proteasome inhibitor approved for patients with relapsed and/or refractory multiple myeloma (RRMM) who failed ≥ 1 prior lines of therapy. We performed a systematic review of Carf literature with meta-analysis to determine the efficacy and safety in RRMM patients.MethodsBased on literature search, we included a total of 14 eligible phase I/II, phase II and phase III Carf based clinical trials. The cumulative incidence and odds ratios (OR) were calculated with random effect model, using ‘’R’’ software with metaphor package.Results2906 evaluable RRMM patients from published clinical trials included. The pooled overall response rate (ORR) was 45% (95% CI: 29–62). The pooled clinical benefit rate (CBR) was 56% (95% CI: 41–71). OR from 3 randomized clinical trials showed that Carf significantly improved ORR and CBR compared to control groups (OR 2.4, P < 0.0001; 2.02, P = 0.0007, respectively). Subgroup analysis showed significantly better ORR (P < 0.0001) and CBR (P < 0.001) with combination regimens compared to monotherapy. Response was significantly higher with high dose of Carf (>20/27 mg/m2) compared to standard dose (ORR 65% vs. 35%, P = 0.03). Compared to control group, the OR of developing cardiotoxicity (P = 0.002) and hypertension (P < 0.0001) were significantly higher with Carf, while no difference in peripheral neuropathy (P = 0.28).ConclusionsCarf produces significantly better responses with acceptable safety profile in RRMM patients. Combination regimens and higher dose Carf offers better response with no significant extra toxicity. Its efficacy is regardless of cytogenetics or disease stage. Incidences of cardiotoxicity and hypertension seem higher with Carf.

P-320 - Comparative effects of bortezomib and carfilzomib in co-culture of human neural progenitor cells and leukemia cells

Inhibition of the proteasomal system as a cancer treatment is a powerful strategy. After the FDA approval of the first proteasome inhibitor, bortezomib, proteasome inhibition proved itself as an effective target for treating hematological cancers. However, neurotoxicity is one of the most important side effects of bortezomib that limits its use in clinical practice. Carfilzomib is a second-generation proteasome inhibitor and appears to a have better neurotoxic profile than bortezomib. Despite the higher prevalence of bortezomib neurotoxicity, mechanisms underlying this difference are not well understood. In this study, we comparatively investigated the effects of bortezomib and carfilzomib on human neural progenitor and leukemia cells in a co-culture model. Ubiquitinated and carbonylated proteins, and HSPs levels are evaluated for understanding the stress response. Apoptosis was analyzed with Annexin V/PI staining by flow cytometry. Also, PARP and caspase 9 levels were determined. Additionally, cytoskeletal proteins changes were assessed with confocal microscopy. Our preliminary results showed that bortezomib and carfilzomib affected stress response and induced apoptosis in a different manner. Also, bortezomib caused more severe alterations in cytoskeletal proteins.

Abstract OT3-07-01: Ixazomib in combination with carboplatin in pretreated women with advanced triple negative breast cancer, an ongoing phase I/II trial (AGMT MBC-10 trial)

Abstract Background:Triple-negative breast cancer (TNBC) comprises a heterogeneous group of diseases which are generally associated with poor prognosis. Recently, the PARP inhibitor olaparib was approved as first targeted treatment beyond antiVEGF therapy for the BRCA1/2 mutated subgroup of TNBC. However, cytotoxic agents still remain the mainstay of treatment for this breast cancer subtype. Ixazomib is a selective and reversible inhibitor of the proteasome, which has been mainly investigated as treatment of multiple myeloma. In a preclinical cell line model for TNBC the first-generation proteasome inhibitor bortezomib showed synergistic efficacy with cisplatin. Clinical data are available for carboplatin plus bortezomib in metastatic ovarian and lung cancers showing remarkable antitumor activity (47% and 38% response rate, respectively) and good tolerability. In solid tumors cytotoxic effect of proteasome inhibitors is thought to be mediated through different mechanisms: (1) Inhibition of the Fanconi Anemia and BRCA1 DNA repair mechanism (2) Inhibition of p53 degradation (3) Inhibition of NF-kappa B signaling cascade. Based on this evidence, the phase I/II MBC-10 trial will evaluate the toxicity profile and efficacy of the oral second-generation proteasome inhibitor ixazomib in combination with carboplatin in patients with advanced TNBC. Trial Design: Patients with metastatic TNBC pretreated with at least one prior line of chemotherapy for advanced disease with a confirmed disease progression and measurable disease are eligible for this study.Patients will receive ixazomib in combination with carboplatin on days 1, 8, and 15 in a 28-day cycle. The phase I part of this study uses an alternate dose escalation accelerated titration design. After establishing the maximum tolerated dose (MTD), accrual continues to evaluate the efficacy and safety of the combination (phase II, including 41 evaluable patients). All patients will continue on study drugs until disease progression, unacceptable toxicity or discontinuation for any other reason. Primary endpoint of the phase II is overall response rate, secondary endpoints include safety profile, progression-free survival and quality of life. The MBC-10 trial is accompanied by a broad biomarker program investigating predictive biomarkers for treatment response and potential resistance mechanisms to the investigational drug combination. This trial is open for patient enrollment since November 2016 in six Austrian cancer centers. Accrual is planned to be completed within two years. ClinicalTrials.gov Identifier: NCT02993094 Citation Format: Rinnerthaler G, Gampenrieder SP, Petzer A, Burgstaller S, Voskova D, Rossmann D, Balic M, Egle D, Rumpold H, Singer CF, Petru E, Melchardt T, Ulmer H, Mlineritsch B, Greil R. Ixazomib in combination with carboplatin in pretreated women with advanced triple negative breast cancer, an ongoing phase I/II trial (AGMT MBC-10 trial) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr OT3-07-01.

Discovery of Natural Proteasome Inhibitors as Novel Anticancer Therapeutics: Current Status and Perspectives

Natural products serve as a main resource for drug discovery. The ubiquitin-proteasome system (UPS) is one of the primary intracellular protein degradation systems, which is responsible for the degradation of most short-lived, mis-folded and aged proteins. The proteasome is a validated target for cancer treatment, since cancer cells are more reliant on high levels of proteasome activity to maintain the dynamic protein homeostasis required for enhanced metabolism and unrestricted proliferation. Encouraged by success of bortezomib in the treatment of multiple myeloma, several second-generation proteasome inhibitors have been developed based on natural resources, and are being tested in various clinical settings. In this paper, we reviewed the most widely investigated proteasome inhibitors, including their natural product origins, compound-discovery and optimization, as well as their current status in both preclinical and clinical studies.

Phase 1/2 Trial of Carfilzomib Plus High-Dose Melphalan Preparative Regimen for Salvage Autologous Hematopoietic Cell Transplantation Followed by Maintenance Carfilzomib in Patients with Relapsed/Refractory Multiple Myeloma

We performed a phase 1/2 trial to investigate the safety and activity of the second-generation proteasome inhibitor Carfilzomib (K) on days -3/-2 in combination with melphalan 200 mg/m2 (MEL200) on day -2 (K-MEL) in patients with relapsed multiple myeloma (MM) undergoing autologous hematopoietic cell transplantation (phases 1 and 2). Patients without progression received 12 cycles of K maintenance at 36 mg/m2 days 1, 8, and 15 (schedule A) or days 1, 2, 15, and 16 (schedule B), with patients being treated for 2 cycles in each schedule and on the patient-preferred schedule for the remaining cycles (phase 2). The patients had received a median of 3 previous lines of therapy, 56% had undergone previous AHCT, and 51% had received previous K therapy. During phase 1 (n = 15), the maximum tolerated dose of K in combination with MEL200 was not reached, so the maximum tested dose of 27 mg/m2 (on day -3) and 56 mg/m2 (on day -2) was used in phase 2. The rate of very good partial response after K-MEL therapy (n = 44) was 59.2%, compared with 13.7% before K-MEL therapy. Among patients starting maintenance therapy (n = 27), 12-month progression-free survival was 66.7% and 12-month overall survival was 88.1%. There was no strong patient preference for either schedule. Two patients discontinued maintenance due to toxicity. K-MEL followed by K maintenance is safe and active salvage therapy in patients with MM.

Carfilzomib.

Carfilzomib (CFZ) is a potent, second-generation proteasome inhibitor (PI), with significant activity as a single agent and in combination with other antimyeloma agents in patients with relapsed or refractory multiple myeloma (RRMM). CFZ binds selectively and irreversibly to its target and leads to antiproliferative and proapoptotic effects on cancer cells. This irreversible inhibition is dose- and time-dependent in vitro and in vivo. CFZ as monotherapy and in combination with other antimyeloma agents (e.g., as CFZ and dexamethasone [Kd]) achieved very good responses, progression-free survival (PFS) and overall survival (OS). In several ongoing studies, CFZ is being investigated in triplet and quadruplet schedules of CFZ, lenalidomide and dexamethasone (KRd), CFZ, cyclophosphamide, dexamethasone (KCd) and with antibodies, like elotuzumab or daratumumab. The multitude of completed and ongoing studies confirmed a tolerable safety profile of CFZ, a significantly lower incidence of neuropathy compared to bortezomib (BTZ) and a slightly higher incidence of cardiotoxicity, which is closely observed and precautions taken to avoid them as best as possible. In July 2012, the US Food and Drug Administration (FDA) approved CFZ as a single agent for RRMM patients with disease progression after two prior therapies, including BTZ and immunomodulatory drugs (IMiDs). The combination of KRd and Kd followed, being approved by both FDA and European Medicines Agency (EMA) in 2015 and 2016, respectively. Moreover, CFZ is being evaluated in patients with newly diagnosed MM (NDMM), in high-risk smoldering MM and for maintenance approaches.

Anti-leukemic activity of bortezomib and carfilzomib on B-cell precursor ALL cell lines.

Prognosis of childhood acute lymphoblastic leukemia (ALL) has been dramatically improved. However, prognosis of the cases refractory to primary therapy is still poor. Recent phase 2 study on the efficacy of combination chemotherapy with bortezomib (BTZ), a proteasome inhibitor, for refractory childhood ALL demonstrated favorable clinical outcomes. However, septic death was observed in over 10% of patients, indicating the necessity of biomarkers that could predict BTZ sensitivity. We investigated in vitro BTZ sensitivity in a large panel of ALL cell lines that acted as a model system for refractory ALL, and found that Philadelphia chromosome-positive (Ph+) ALL, IKZF1 deletion, and biallelic loss of CDKN2A were associated with favorable response. Even in Ph-negative ALL cell lines, IKZF1 deletion and bilallelic loss of CDKN2A were independently associated with higher BTZ sensitivity. BTZ showed only marginal cross-resistance to four representative chemotherapeutic agents (vincristine, dexamethasone, l-asparaginase, and daunorubicin) in B-cell precursor-ALL cell lines. To improve the efficacy and safety of proteasome inhibitor combination chemotherapy, we also analyzed the anti-leukemic activity of carfilzomib (CFZ), a second-generation proteasome inhibitor, as a substitute for BTZ. CFZ showed significantly higher activity than BTZ in the majority of ALL cell lines except for the P-glycoprotein-positive t(17;19) ALL cell lines, and IKZF1 deletion was also associated with a favorable response to CFZ treatment. P-glycoprotein inhibitors effectively restored the sensitivity to CFZ, but not BTZ, in P-glycoprotein-positive t(17;19) ALL cell lines. P-glycoprotein overexpressing ALL cell line showed a CFZ-specific resistance, while knockout of P-glycoprotein by genome editing with a CRISPR/Cas9 system sensitized P-glycoprotein-positive t(17;19) ALL cell line to CFZ. These observations suggested that IKZF1 deletion could be a useful biomarker to predict good sensitivity to CFZ and BTZ, and that CFZ combination chemotherapy may be a new therapeutic option with higher anti-leukemic activity for refractory ALL that contain P-glycoprotein-negative leukemia cells.

Improving the efficacy of proteasome inhibitors in the treatment of renal cell carcinoma by combination with the human immunodeficiency virus (HIV)-protease inhibitors lopinavir or nelfinavir.

To assess the potential of second-generation proteasome inhibition by carfilzomib and its combination with the human immunodeficiency virus (HIV) protease inhibitors (HIV-PIs) lopinavir and nelfinavir in vitro for improved treatment of clear cell renal cell cancer (ccRCC). Cytotoxicity, reactive oxygen species (ROS) production, and unfolded protein response (UPR) activation of proteasome inhibitors, HIV-PIs, and their combination were assessed in three cell lines and primary cells derived from three ccRCC tumours by MTS assay, flow cytometry, quantitative reverse transcriptase-polymerase chain reaction and western blot, respectively. Proteasome activity was determined by activity based probes. Flow cytometry was used to assess apoptosis by annexin V/propidium iodide assay and ATP-binding cassette sub-family B member 1 (ABCB1) activity by MitoTracker™ Green FM efflux assay (Thermo Fisher Scientific, MA, USA). Lopinavir and nelfinavir significantly increased the cytotoxic effect of carfilzomib in all cell lines and primary cells. ABCB1 efflux pump inhibition, induction of ROS production, and UPR pre-activation by lopinavir were identified as underlying mechanisms of this strong synergistic effect. Combined treatment led to unresolved protein stress, increased activation of pro-apoptotic UPR pathway, and a significant increase in apoptosis. The combination of the proteasome inhibitor carfilzomib and the HIV-PIs lopinavir and nelfinavir has a strong synergistic cytotoxic activity against ccRCCin vitro at therapeutically relevant drug concentrations. This effect is most likely explained by synergistic UPR triggering and ABCB1-modulation caused by HIV-PIs. Our findings suggest that combined treatment of second-generation proteasome inhibitors and HIV-PIs should be investigated in patients with metastatic RCC within a clinical trial.

An overview of the role of carfilzomib in the treatment of multiple myeloma

ABSTRACTIntroduction: Carfilzomib is a second-generation proteasome inhibitor that binds selectively and irreversibly with the chymotrypsin-like site of the proteolytic core. Its initial approval by the Food and Drug Administration, as monotherapy for relapsed/refractory multiple myeloma (RR-MM), followed soon by a global authorization of its combination with dexamethasone or with lenalidomide plus dexamethasone for the treatment of RR-MM after 1–3 prior lines. In order to optimize its administration, carfilzomib is currently examined in different doses and regimens in relapsed/refractory as well as in newly diagnosed myeloma.Areas covered: This review will focus on the introduction of carfilzomib as an effective anti-myeloma treatment, describing the evolution of the drug from its pre-clinical development to its established use by phase III clinical trials. Based on the latest evidence, we will present its mechanism of action, its efficacy and its toxicity profile on treated myeloma patients and we will try to reply to all raised concerns about its current use.Expert opinion: Either alone or in combination with other agents, carfilzomib seems to be an effective and safe therapeutic option for MM management. Results of ongoing trials are expected to update its application, even at an earlier level of the disease course.