Combination Of Carfilzomib Research Articles

Sorafenib and Carfilzomib Synergistically Inhibit the Proliferation, Survival, and Metastasis of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common and deadly human cancers. The 5-year survival rate is very low. Unfortunately, there are few efficacious therapeutic options. Until recently, Sorafenib has been the only available systemic drug for advanced HCC. However, it has very limited survival benefits, and new therapies are urgently needed. In this study, we investigated the anti-HCC activity of carfilzomib, a second-generation, irreversible proteasome inhibitor, as a single agent and in combination with sorafenib. In vitro, we found that carfilzomib has moderate anticancer activity toward liver cancer cells, but strongly enhances the ability of sorafenib to suppress HCC cell growth, proliferation, migration, invasion, and survival. Remarkably, the drug combination exhibits even more potent antitumor activity when tested in animal tumor models. Mechanistically, the combined treatment activates caspase-dependent and endoplasmic reticulum stress/CHOP-mediated apoptotic pathways, and suppresses epithelial-mesenchymal transition. In conclusion, our results demonstrate that the combination of carfilzomib and sorafenib has synergistic antitumor activities against HCC, providing a potential therapeutic strategy to improve the mortality and morbidity of HCC patients.

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.

Co-Targeting of BCL2 with Venetoclax and MCL1 with S63845 Is Synthetically Lethal In Vivo in Relapsed Refractory Mantle Cell Lymphoma with Complex Karyotype Changes

IntroductionMantle cell lymphoma (MCL) is an aggressive subtype of B-cell non-Hodgkin lymphomas characterized by (over)expression of BCL2 and good sensitivity to a small molecule BCL2 inhibitor venetoclax. In the present study we analyzed molecular mechanisms of venetoclax resistance in MCL cells, and tested strategies to overcome it based on concurrent targeting of BCL2 a MCL1.MethodsCell death was determined by flow cytometry using Annexin-V/PI staining. Establishment of MCL cell clones with knock-down or transgenic overexpression of MCL1, BIM and NOXA, western blotting, immunohistochemistry of formalin-fixed paraffin-embedded tissue sections, and immunoprecipitation experiments were carried out as previously described (Klanova et al, Clin Cancer Res, 2016). All PDXs were derived in our laboratory from patients with relapsed MCL. All PDX were confirmed by NGS to keep majority of somatic mutations with the primary MCL cells from which they were derived. Samples were sequenced using SureSelectXT Human All Exon V6+UTR (Agilent Technologies, Santa Clara, CA) on the NextSeq 500 (Illumina, San Diego, CA) instrument according to manufacturer's protocols. Experimental therapies were implemented using NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice purchased from Jackson Laboratory (Bar Harbor, Maine, USA). Therapy was initiated when all mice developed palpable subcutaneous tumors (= day 1, D1). Venetoclax (VTX) and S63845 were from MedchemExpress, carfilzomib (CFZ) was from Charles University General hospital pharmacy. Carfilzomib (4 mg / kg) was administered intravenously (IV) on days 1 and 6. Venetoclax (40 mg / kg) was given by oral gavage on days 1, 2, 3, 6 and 7. S63845 (25 mg / kg) was administered IV on days 1, 2, 3, 6 and 7. Tumor volumes were calculated using the following formula: π / 6 × tumor length × width × height.ResultsBy transgenic overexpression or shRNA-mediated knock-down we confirmed key roles of proapoptotic proteins BIM and NOXA in mediating venetoclax-induced cell death in MCL. We demonstrated that both BIM and NOXA are differentially expressed between MCL cell lines on one side, and primary MCL cells and patient-derived xenograft (PDX) cells on the other side. First, NOXA protein is significantly overexpressed in most MCL cell lines. Second, biallelic deletions of BIM harbored by three commonly used MCL cell lines (JEKO-1, MINO and Z138), and previously reported to be present in approx. 30% of MCL patients, were not found in primary MCL cells. As a consequence, vast majority of the in vitro data was implemented on venetoclax-sensitive cell lines HBL2 and MAVER-1, whose patterns of expression of BCL2, MCL1, BIM and NOXA are similar to primary MCL cells. We demonstrated that MCL1, another key anti-apoptotic protein, plays an essential role in mediating resistance to venetoclax. First, MCL1 functions as a buffer for BIM released from BCL2 upon binding of venetoclax thereby preventing activation of BAX and induction of apoptosis. Second, marked upregulation of MCL1 protein was associated with acquired resistance to venetoclax in two most sensitive MCL cell lines HBL2 and MAVER-1. Based on the in vitro data we proposed two experimental treatment strategies that co-targeted MCL1 (along with inhibition of BCL2 with venetoclax): a direct blockage with a highly specific small molecule MCL1 inhibitor S63845, and an indirect blockage achieved by proteasome inhibitor carfilzomib that upregulates the proapoptotic protein NOXA that specifically binds and blocks MCL1. The combination of venetoclax and S63845 demonstrated synthetic lethality in vivo inducing the longest “remissions” of MCL bearing mice (i.e. temporary disappearance of subcutaneous MCL tumors) using a panel of four different PDXs derived from patients with relapsed / refractory MCL with complex karyotype changes (Figure 1). The combination of carfilzomib and venetoclax was far less effective, and at the same time more toxic suggesting functional blockage of MCL1 induced by overexpressed NOXA is either incomplete or insufficient.ConclusionsOur data strongly support investigation of venetoclax in combination with S63845 as an innovative proapoptotic treatment strategy for chemoresistant MCL patients with adverse cytogenetics in the clinical grounds. [Display omitted] DisclosuresTrněný:Janssen: Membership on an entity's Board of Directors or advisory committees, Other: Advisory board; Gilead: Honoraria; Morphosys: Membership on an entity's Board of Directors or advisory committees, Other: Advisory board; Abbvie: Honoraria, Research Funding; F. Hoffman-La Roche Ltd: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory board, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory board; Sandoz: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory board; Incyte: Membership on an entity's Board of Directors or advisory committees, Other: Advisory board.

Synergy between FLT3-ITD and p53 Haploinsufficiency or Loss in the Development of Acute Myeloid Leukemia

FLT3-ITD (internal tandem duplication) is a late event in the pathogenesis of acute myeloid leukemia (AML). Identification of early cooperating events for FLT3 mutations may improve our understanding of the pathogenesis of AML and lead to a more efficient treatment and improved outcome for AML patients. p53 alteration can be found in up to 70% of AML patients with complex karyotypes, while studies from our group and others show a relatively low incidence of p53 mutation (generally around 10%) in other AML patients. Dysfunction of p53 pathway resulting from overexpressed MDM2/MDM4 is more often found than p53 mutations in patients with de novo AML. An early mouse study identified the FLT3 gene to be preferentially mutated by insertional mutagenesis in p53 knock-out but not in p53 wild-type tumors. Importantly, p53 mutation appears to be an early event in the pathogenesis of AML. In this study, we investigated whether p53 haploinsufficiency or loss cooperates with FLT3-ITD in the induction of AML.To this end, we crossed FLT3-ITD knock-in mice with p53 knockout mice to generate mice harboring both ITD/ITD and p53 knockout mutations. ITD/ITD; p53+/- (FLT3-ITD homozygous and p53 heterozygous) mice became moribund much faster than mice with ITD/ITD alone or p53+/- alone. The median survival of ITD/ITD;p53+/- mice (n=69) was 313 days, which was significantly shorter than that of ITD/ITD mice (littermates of ITD/ITD; p53+/- mice, 583 days, n=16), p53+/- mice (521 days, n=30), and WT mice (862 days, n=10) (p<0.0001). Interestingly, the median survival ITD/ITD;p53-/- mice (FLT3-ITD homozygous and p53 homozygous, littermates of ITD/ITD; p53+/- mice, n=11) was further reduced (125 days), and significantly shorter than ITD/ITD; p53+/- mice and p53-/- mice (148 days, n=15) (p<0.05). Moreover, cooperation of FLT3-ITD and p53 inactivation also significantly altered disease spectrum in mice. While AML was observed only in 25% ITD/ITD mice and p53+/- mice mainly developed solid tumors, 88% ITD/ITD; p53+/- mice developed acute leukemia including AML (58%). The incidence of AML was further increased in ITD/ITD; p53-/- mice (73%, 8/11). All ITD/ITD; p53-/- mice developed acute leukemia, while 72% p53-/- mice developed lymphoma/lymphoblastic leukemia. Interestingly, >50% ITD/ITD; p53-/- mice and ITD/ITD; p53+/- mice with acute leukemia demonstrated a bi-clone disease, with co-existence of AML and ALL. Unexpectedly, all analyzed murine AMLs with complete or heterozygous loss of p53 (n=9) showed normal karyotypes. p53 haploinsufficiency or loss did not increase self-renewal of hematopoietic stem/progenitor cells as defined by serial replanting assays in vitro and limiting dilution transplantation of leukemic cells in vivo. However, animals with only AML in the ITD/ITD; p53+/- group showed a significant increase of CMPs in comparison with ITD/ITD mice with CMML, P53+/- and WT mice, e.g. a 66-fold increase of CMPs in #1376 mouse compared with WT mice. This suggests that the block of differentiation from CMPs to GMPs may have contributed to the development of AML.P53 haploinsufficiency or loss reduced the sensitivity of murine FLT3-ITD leukemia to crenolanib in vitro (IC50: 313.3nM, 461.4nM, and 185.8nM for ITD/ITD;p53+/-, ITD/ITD;p53-/-, and ITD/ITD leukemia, respectively), but not their sensitivity to midostaurin (IC50: 117.3nM, 110.2nM, and 201.3nM). To develop an efficient therapy for p53-mutated AML, we firstly tried to combine FLT3 inhibitors and MDM2 antagonist idasanutlin, FLT3 inhibitor and Bcl2 inhibitor venetoclax. Unfortunately, these did not enhance induction of apoptosis in leukemic cells from ITD/ITD;p53+/- mice. However, exposure to the proteasome inhibitor carfilzomib had a strong cytotoxic effect against ITD/ITD; p53+/- and ITD/ITD;p53-/-, leukemic cells (IC50: 5.2nM and 17.1nM vs. 6.8nM for ITD/ITD leukemia). In addition, the combination of carfilzomib with midostaurin showed an additive cytotoxicity in murine ITD/ITD; p53+/- leukemia as well as in primary leukemic cells from most patients with AML (n=10).Taken together, our data indicate a strong cooperating effect of FLT3-ITD and p53 haploinsufficiency or loss in the induction of AML and ALL. Our data emphasize more careful analysis of p53 deregulation in AML. Targeting FLT3 in combination with drugs working independently of p53 status, such as proteasome inhibitor carfilzomib, might improve outcomes of AML patients. DisclosuresGanser:Novartis: Membership on an entity's Board of Directors or advisory committees.

MCRN¯003/MYX·1: A Single Arm Phase II Study of High-Dose Weekly Carfilzomib Plus Cyclophosphamide and Dexamethasone in the Treatment of Relapsed Multiple Myeloma after 1-3 Prior Therapies

Background: Carfilzomib, a second generation proteosome inhibitor, is effective in the treatment of relapsed and refractory multiple myeloma (RRMM). Recent phase II and phase III trials have demonstrated the efficacy of weekly dosing strategies. The aim of this study was to examine high dose once weekly carfilzomib in combination with weekly dexamethasone and low dose weekly cyclophosphamide (wCCD) in RRMM. It was hypothesized that this may offer a potent yet convenient and more financially viable triplet-based treatment option than existing combinations.Methods: The MCRN-003/MYX.1 multi-centre single arm phase II clinical trial is run through the Myeloma Canada Research Network (MCRN) with support from the Canadian Cancer Trials Group (CCTG). Patients who had at least one but not more than three prior lines of therapy and who did not have proteosome inhibitor (PI) refractory disease were eligible. Treatment consists of carfilzomib (20 mg/m2 day 1 of first cycle then escalated to 70 mg/m2 for all subsequent doses) given on days 1, 8, and 15 of a 28-day cycle, plus weekly oral dexamethasone 40 mg and cyclophosphamide 300 mg/m2 capped at 500 mg on days 1, 8, 15 and 22. Treatment continues until progression or intolerance, except for cyclophosphamide which is discontinued after 12 cycles. The total sample size of 76 patients includes a 6 patient lead-in phase where safety at 70 mg/m2 was evaluated. The primary objective was to observe an overall response rate (ORR) ≥ 80% after 4 cycles of protocol therapy. Secondary endpoints include safety, toxicity, kinetics of and maximal response depth and overall survival. This analysis is based on the locked data base of 2018 July 13.Results: Of the 76 patients accrued 1 was subsequently determined to be ineligible on the basis of bortezomib refractory disease, and 1 did not receive any protocol therapy due to a cardiac event occurring post-study registration but prior to treatment commencement. All patients who received therapy were included in the analysis as per protocol inclusive of the bortezomib exposed patient. Among these 75 patients, median age was 66 years with 33% being > 70 years of age. Thirty-seven percent were female. Thirty-nine percent received 1 prior line, 44% received 2 prior lines and 17% received 3 prior lines of therapy. High risk cytogenetics [(t4;14), t(14;16) and del P53] were identified in 32%. Twenty percent had ISS stage III disease and 11% had R-ISS stage III disease. Prior PI and immunomodulatory drug exposure was noted in 87% and 81% respectively.Within the first 4 cycles of therapy 84% (95% CI, 76-92%) of patients achieved PR or better, with ≥ VGPR achieved in 52% and ≥ CR in 9% (table 1, p = 0.0006). There was a trend toward a better ORR after 4 cycles based on the presence or absence of high-risk cytogenetics (75% vs 94% respectively, p = 0.051) not meeting statistical significance. The median duration of follow-up at the time of data analysis was 13.9 months (range 0.2 to 22.8 months). 18 patients have died with an estimated 1-year OS of 80%. The cause of death as assessed by the investigator was myeloma in 13 patients with 3 dying from a cause possibly or probably related to the study intervention.During the first 4 cycles of treatment, non-hematologic toxicity ≥ grade 3 occurred in 33% of patients; most commonly infection (16%) and fatigue (7%). Grade 3/4 anemia was observed in 17%, thrombocytopenia in 33% and neutropenia in 20%. Grade 3 or greater hypertension was seen in 4%, dyspnea in 1%, pulmonary edema in 1% and thrombotic microangiopathy in 4%; all resolved with no long-term sequelae. To date 37 (49%) patients have discontinued carfilzomib, 11 due to toxicity and 16 due to disease progression.Conclusion: This prospective phase II study demonstrates that wCCD is a safe and effective regimen in the treatment of RRMM. The study met its primary endpoint demonstrating a ≥ 80% ORR after 4 cycles of therapy. These results compare favourably to published phase III data examining weekly carfilzomib and dexamethasone as well as the established twice-weekly dosing strategies. This regimen will be a useful triplet-based option for RRMM especially in patients refractory to immunomodulatory agents who would otherwise be ineligible for the carfilzomib-lenalidomide-dexamethasone combination. [Display omitted] DisclosuresVenner:Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Amgen: Honoraria; Takeda: Honoraria. Leblanc:Celgene Canada: Membership on an entity's Board of Directors or advisory committees; Janssen Inc.: Membership on an entity's Board of Directors or advisory committees; Amgen Canada: Membership on an entity's Board of Directors or advisory committees; Takeda Canada: Membership on an entity's Board of Directors or advisory committees. Sandhu:Celgene: Honoraria; Janssen: Honoraria; Amgen: Honoraria; Novartis: Honoraria; Bioverativ: Honoraria. White:Amgen, Celgene, Janssen, Takeda: Honoraria. Chen:Amgen: Honoraria. Louzada:Celgene: Honoraria; Janssen: Honoraria; amgen: Honoraria; pfizer: Honoraria. Hay:Amgen: Research Funding; Novartis: Research Funding; Janssen: Research Funding; Roche: Research Funding; Seattle Genetics: Research Funding; Kite: Research Funding.

Once Weekly Versus Twice Weekly Carfilzomib in Combination with Cyclophosphamide and Dexamethasone in Newly Diagnosed Multiple Myeloma: A Pooled Analysis of Two Phase 1/2 Studies

Once Weekly Versus Twice Weekly Carfilzomib in Combination with Cyclophosphamide and Dexamethasone in Newly Diagnosed Multiple Myeloma: A Pooled Analysis of Two Phase 1/2 Studies

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.

Carfilzomib Weekly 20/56mg/m², Lenalidomide and Dexamethasone for Early Relapsed Refractory Multiple Myeloma

Background. Triplet-based lenalidomide plus dexamethasone combinations have become the new standard of care for early RRMM, including with carfilzomib (KRd). In Aspire phase 3 trial for registration, carfilzomib was given on a dose dense twice a week but not dose intense 20/27 mg/m² dose concentration. In parallel, several studies seem to demonstrate that dose intensity, with increased dose concentration of carfilzomib, could compensate for a less dose dense regimen with carfilzomib weekly. We hypothesized that weekly carfilzomib regimen of KRd will improve time on treatment and quality of life and thus PFS and possibly OS. We aimed to evaluate the efficacy and toxicity of carfilzomib weekly plus lenalidomide/dexamethasone regimen in early RRMM.Methods. 28 patients were prospectively recruited. Carfilzomib was administered as a 30-minute infusion on days 1,8,15 (at 20mg/m2 on day 1 of cycle 1; then 56mg/m2 thereafter). Lenalidomide (25mg/days) was given 21/28. Dexamethasone was administered weekly.Results. With a median follow-up of 25 months, 42% relapsed and 17% died. Importantly, 16/28 (57%) are still on treatment. The median number of cycles was 18, with 32% of patients treated more than 20 cycles. Only 4 patients stopped weekly KRd for safety issues. ORR and CBR was 85.7% and 89.3%, whom 43% ≥CR; with a median DOR of 13 months and 43% having more than 18 months. 7 patients had negative MRD at 10-5 and normalized PET CT. The projected PFS, EFS and OS at 30 months were 56%, 43% and 82%, respectively. Hematologic and non-hematologic adverse events ≥ grade 3 were reported in 57% and 36%, respectively. Adverse events ≥ grade 3 seen in ≥10% of patients were neutropenia, thrombocytopenia, vomiting and pyrexia.Conclusion. KRd weekly at 20/56mg/m2 is effective and safe to early RRMM patients, provides improved safety profile and QOL to patients allowing treating patients until progression. Further studies are warranted to confirm this data on a larger early RRMM population and validate the ability to use dose intensity to improve the dose density approach using carfilzomib combination. DisclosuresLeleu:BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Mundipharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

The Status of Multiple Myeloma in Colombia: First Report of the Colombian Registry for Hemato-Oncological Diseases (RENEHOC). Asociacion Colombiana De Hematologia y Oncologia (ACHO)

IntroductionMultiple myeloma (MM) has characteristics that influence the prognosis and are useful to make decisions regarding treatment. Diagnosis and treatment patterns of hematological malignancies have not been described in Colombia and having access to this regional information could guide public health policies and help design clinical trials. The Colombian Association of Hematology and Oncology (ACHO) designed a registry for Hemato-Oncological diseases to fill this information gap. This is the first report of RENEHOC focusing on MM.MethodsWe conducted an ambispective multi-centric cohort study of clinical records available from patients with MM that were diagnosed in the most populated regions of Colombia. We collected demographic, diagnostic, clinical and treatment data at different time points from diagnosis to the most current follow up. The computerized de-identified data was consolidated and analyzed using basic statistical measures of central tendency and dispersion. The most current follow up included data available from last clinic visit, loss of follow up, death or withdrawal of consent. This study was approved by local and centralized institutional review board.ResultsWe analyzed data from 206 MM patients (pts). Average age at diagnosis was 62.9 (SD 10.2) years and 51.4% of pts were male. Most common symptoms at diagnosis were bone pain (74.2%), anemia related (58.2%) and pathological fractures (45.1%). Mean time from symptoms to diagnosis was 7.3 (Range 1-44) months. Baseline mean hemoglobin, creatinine and calcium serum levels were 10.5 g/dL, 1.58 mg/dL and 9.2 mg/dL respectively. At diagnosis, 22% of patients presented with renal failure and 30% required dialysis.Immunoglobulin (Ig) subtype distribution was: IgG 50.6%, IgA 20.6%, only light chain secretion (61% Kappa) and IgM 1.1%. In 11.6% all Igs were low. Most pts were diagnosed at advanced stages (64.5% Durie-Salmon III, and 37% and 47% International Staging System II and III respectively). Molecular prognostic characterization was performed only in 39 patients (18.4%).Four patients were considered too fragile for active treatment. The most common initial treatment was triple combinations that included Bortezomib (84% of patients). Among those, CyBorD was the most frequently used first line therapy (58.3%), followed by VTD (14.6%). Mean number of cycles was 4.8 (SD 2.5). Overall Response Rate (ORR) 74.7% was observed. Only 40% of patients treated with intense induction therapy received autologous transplant. Response to treatment including transplant are shown in table 1. Second and further lines of treatment were highly variable. 18 patients were treated with a second autologous transplantation consolidating 2nd line therapy. 12 patients received Carfilzomib combinations, all of them in 2nd line. 5 patients were treated with Daratumumab, all >2nd line. Mean follow-up time was 33.53 (SD 3.6) months. After a median follow up of 27 months we observed in our cohort an overall survival of 82% and 68% of patients progression-free. .ConclusionsThis first report focusing on MM highlights important differences on patient's characteristics and treatment in Colombian patients. This includes and treatment initiation due to insurance barriers; high risk clinical characteristics and complications upon diagnosis and low rate of transplantation in patients otherwise eligible for this procedure. Rates of response to induction seem slightly lower than reported (CB Reeder 2009). However, overall survival and progression-free survival met the expectations for this type of patients. The development of country specific databases may influence the design of policies, allocation and use of resources and improve patient outcomes. Only cooperative efforts like the one spearheaded by RENEHOC can achieve those goals. DisclosuresNo relevant conflicts of interest to declare.

Montelukast enhances cytocidal effects of carfilzomib in multiple myeloma by inhibiting mTOR pathway

ABSTRACT Montelukast is an anti-asthmatic medication, and has recently showed its inhibitory effects on the proliferation of cancers. The purpose of this study was to identify the cytotoxic effects of montelukast on multiple myeloma (MM) cells and the combination effects of montelukast and carfilzomib in the treatment of MM. Results revealed that montelukast induced a dose- and time-dependent cytotoxicity in MM cells lines and significantly suppressed the colony formation of myeloma cells. Furthermore, montelukast enhanced the cytotoxicity of carfilzomib in MM cell lines. This anti-tumor effect was associated with decreased c-Myc via the inhibition of mTOR signaling pathway. Moreover, the combination of montelukast and carfilzomib induced apoptosis of myeloma cells effectively, even in the presence of bone marrow stromal cells (BMSCs). It is more important to note that the co-treatment exhibited similar cytocidal effects in carfilzomib-resistant cell lines (U266R and 8226R). In addition, the combined effects were noted in two MM xenograft mice models and 7 cases of human CD138+ myeloma cells (4 newly diagnosed cases and 3 relapsed cases) with no cytotoxicity on peripheral blood mononuclear cells (PBMCs) from 5 healthy donors. Our data suggested that montelukast enhanced the cytotoxicity of carfilzomib in both carfilzomib-sensitive and carfilzomib-resistant MM cell lines. These findings may facilitate the development of therapeutic strategies and provide a promising therapeutic combination regimen for the treatment of refractory myeloma.

PRM46 - MEASURING AND ANALYSING THE MATURITY OF OVERALL SURVIVAL DATA FOR TREATMENTS FOR RELAPSED OR REFRACTORY MULTIPLE MYELOMA

Maturity of survival data is a key consideration in the assessment of the value of oncology drugs based on clinical trial evidence. Where there is a hypothesis of a cure fraction or other sources of non-proportional hazards, understanding the quantity and quality of information within Kaplan-Meier tails becomes particularly important in the analysis and interpretation of survival data. Despite this there are limited metrics for and no clear standards applied to data maturity. We calculate indices of data completeness and sensitivity over time for combination therapies in relapsed or refractory multiple myeloma, to demonstrate concepts of data maturity that could be potentially useful. Kaplan-Meier curves for overall survival were sourced from pivotal study publications and published long-term follow-up data for combination therapies licensed by the EMA since 2015 for second-line treatment of multiple myeloma. We digitised survival curves and calculated indices for data completeness and sensitivity for five regimens: elotuzomab; carfilzomib in combination with lenalidomide and dexamethasone and with dexamethasone alone; daratumumab in combination with bortezomib and dexamethasone and with lenalidomide and dexamethasone. Plots of data completeness demonstrate substantial differences in the length of follow-up for which mature data is available. While both daratumumab regimens have generated strong hazard ratios, mature data is available for a much more limited period than other regimens. Plots of data sensitivity show noticeable declines in reliability of information beginning approximately 6 months prior to maximum follow-up for most regimens, with elotuzomab declines beginning somewhat earlier. Indices of data completeness and sensitivity are simple and visually appealing means of measuring and comparing survival data maturity. Use of these indices and development of consensus threshold values that can be applied to them can be a valuable tool in the comparative assessment of oncology therapies.

Abstract 1889: Synergistic effects of glutaminase and proteasome inhibition in multiple myeloma cell lines

Abstract Background: Multiple Myeloma (MM) accounts for approximately 2.1% of all cancer deaths with a 5-year median survival rate of 49.6%. Besides chemotherapy, treatment options include monoclonal antibodies, immunomodulatory agents, and proteasome inhibitors. Carfilzomib is a selective proteasome inhibitor administered intravenously in patients with relapsed or refractory MM. With cancer cell metabolism emerging as a critical regulator of tumor progression, combining carfilzomib with an inhibitor of the metabolic machinery represents a novel therapeutic strategy to prevent MM progression. RP10107 is a potent, and selective glutaminase (GLS-1) inhibitor that demonstrated high potency against mouse (IC50=21.2 nM), rat (IC50=18.2 nM) and human (IC50=26.4 nM) enzymes with selectivity over GLS-2 (&amp;gt;380-fold). The objective of this study was to evaluate the effect of a combination of carfilzomib and RP10107 in MM cell lines. Methods: Glutamate concentrations in MM cell lines (MM-1S and RPMI-8226) following treatment with RP10107 was estimated by LC-MS/MS. Synergism between RP10107 and carfilzomib was determined using different concentrations of the compounds in a 5 x 5 grid. Synergism, additivity, or antagonism was calculated based on the BLISS score. Apoptosis was determined by Annexin V/7AAD staining using a MUSE® Annexin V and dead cell assay kit (Millipore) while the effect on cell cycle was estimated using Guava Cell Cycle Reagent (Millipore). Expression of cleaved PARP and cleaved caspase-8 were determined by Western Blotting. Results: Treatment of MM cell lines with RP10107 resulted in an increased ratio of glutamine to glutamate with a doubling noticed at concentrations as low as 30 nM. A high degree of synergism between RP10107 and carfilzomib was noticed in both the MM cell lines. Addition of 3 nM Carfilzomib to 30 or 500 nM RP10107 resulted in near complete inhibition of RPMI-8226 and MM-1S cell growth, respectively. Additionally, incubation with the combination (1 μM RP10107 + 15 nM Carfilzomib) for 72 h caused a G0/G1 arrest with a corresponding increase in the percent of apoptotic cells (2.5-fold) compared to the individual agents. Incubating RPMI-8226 cells with a combination of RP10107 and Carfilzomib for 48 h resulted in a 2.2- and 13.6-fold increase in expression of cleaved caspase-8 and cleaved PARP, respectively. Conclusions: Addition of RP10107, a glutaminase inhibitor, potentiated Carfilzomib activity in MM cells. Findings provide a rationale for use of the combination in future clinical trials involving MM patients thereby providing a safer and more effective alternative to currently available therapy. Citation Format: Srikant Viswanadha, Satyanarayana Eleswarapu, Kumar V. Penmetsa, Swaroop Vakkalanka. Synergistic effects of glutaminase and proteasome inhibition in multiple myeloma cell lines [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 1889.

Abstract LB-269: IDH2 inhibition enhances proteasome inhibitor responsiveness in hematological malignancies

Abstract The introduction of proteasome inhibitors (PIs) into the clinic has transformed the treatment of patients affected by multiple myeloma (MM) and mantle-cell lymphoma (MCL) establishing new standards of care. Despite these improvements, patients continuously relapse or are intrinsically resistant to PIs. Here, to identify druggable targets that synergize with PIs, we carried out a functional screening in MM cell lines using a short hairpin RNA library targeting 152 cancer driver genes, highly representative of all signaling pathways. The Isocitrate Dehydrogenase 2 (IDH2) gene was identified as a top candidate, showing a synthetic lethal activity with the PI Carfilzomib (CFZ). IDH2 is a NADP(+) dependent mitochondrial enzyme which catalyzes the oxidative decarboxylation of isocitrate to 2-oxoglutarate in the tricarboxylic acid (TCA) cycle. We demonstrated that combinations of the pharmacological IDH2 inhibitor AGI-6780 with FDA approved PIs significantly increased apoptotic cell death in ten MM cell lines, both sensitive and resistant to PIs. Combined treatments triggered synergistic cytotoxicity also in four MCL and in two Burkitt's lymphoma cell lines. Importantly, CFZ/AGI-6780 treatment increased death of primary CD138-positive cells from nine MM patients and exhibited a favorable cytotoxicity profile towards peripheral blood mononucleated cells and bone marrow-derived stromal cells. Mechanistically, CFZ/AGI-6780 combination significantly decreased TCA cycle activity and ATP levels, as a consequence of enhanced IDH2 enzymatic inhibition. In contrast, only a slight increase of mitochondrial reactive oxygen species (ROS) was observed. CFZ treatment reduced the expression of nicotinamide phosphoribosyltransferase (NAMPT), a rate-limiting enzyme required for IDH2 activation through the NAD(+)-dependent deacetylase SIRT3. Consistently, combination of CFZ with either NAMPT (FK866) or SIRT3 (AGK7) inhibitors impaired IDH2 activity and increased MM cell death, thus phenocopying CFZ/AGI-6780 effects and putting the proteasome in a direct link with IDH2 inhibition. Finally, inducible IDH2 knock-down enhanced the therapeutic efficacy of CFZ in a subcutaneous xenograft model of MM, resulting in inhibition of tumor progression and extended survival. In conclusion, our data demonstrate that IDH2 inhibition increases the therapeutic efficacy of PIs, thus providing compelling evidence for treatments with lower and less toxic doses, and broadening the application of PIs to other malignancies. Citation Format: Elisa Bergaggio, Chiara Riganti, Giulia Garaffo, Elisabetta Mereu, Nicoletta Vitale, Cecilia Bandini, Elisa Pellegrino, Paola Omedè, Katia Todoerti, Valentina Audrito, Antonio Rossi, Francesco Bertoni, Silvia Deaglio, Antonino Neri, Antonio Palumbo, Roberto Piva. IDH2 inhibition enhances proteasome inhibitor responsiveness in hematological malignancies [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 LB-269.

Current challenges and opportunities in the management of antibody-mediated rejection in lung transplantation.

There is increasing recognition of the importance of antibody-mediated rejection (AMR) after lung transplantation. The development of donor-specific antibodies, a key feature of AMR, occurs in approximately 30% of lung transplant recipients and is associated with poor posttransplant outcomes. This review highlights recently developed AMR diagnostic criteria in lung transplantation, potential mechanisms that mediate the development of AMR, and discusses current and emerging treatment strategies for this significant, graft-limiting complication. A major advance is the development of consensus guidelines to precisely define AMR amongst lung transplant. Regimens for the treatment of AMR continue to evolve with varying success reported with regards to antibody clearance and improving clinical outcomes. A multimodality treatment approach is common, typically involving a combination of intravenous immune globulin, plasmapheresis, rituximab, and bortezomib or carfilzomib. Recent studies suggest several new agents including tocilizumab, belimumab, daratumumab, plerixafor, and C1 esterase inhibitor as potentially novel and effective therapies to employ in AMR treatment. Despite advancements in the diagnosis of AMR through well defined consensus guidelines, there is limited evidence to guide treatment. Current data suggests that conventional approaches are of suboptimal efficacy, but emerging therapeutic agents with diverse biological mechanisms offer promise for improved AMR treatment.

The Unfolded Protein Response: A Novel Therapeutic Target for Poor Prognostic BRAF Mutant Colorectal Cancer.

BRAFV600E mutations occur in ∼10% of colorectal cancer cases, are associated with poor survival, and have limited responses to BRAF/MEK inhibition with or without EGFR inhibition. There is an unmet need to understand the biology of poor prognostic BRAFMT colorectal cancer. We have used differential gene expression and pathway analyses of untreated stage II and stage III BRAFMT (discovery set: n = 31; validation set: n = 26) colorectal cancer, and an siRNA screen to characterize the biology underpinning the BRAFMT subgroup with poorest outcome. These analyses identified the unfolded protein response (UPR) as a novel and druggable pathway associated with the BRAFMT colorectal cancer subgroup with poorest outcome. We also found that oncogenic BRAF drives endoplasmic reticulum (ER) stress and UPR pathway activation through MEK/ERK. Furthermore, inhibition of GRP78, the master regulator of the UPR, using siRNA or small molecule inhibition, resulted in acute ER stress and apoptosis, in particular in BRAFMT colorectal cancer cells. In addition, dual targeting of protein degradation using combined Carfilzomib (proteasome inhibitor) and ACY-1215 (HDAC6-selective inhibitor) treatment resulted in marked accumulation of protein aggregates, acute ER stress, apoptosis, and therapeutic efficacy in BRAFMT in vitro and xenograft models. Mechanistically, we found that the apoptosis following combined Carfilzomib/ACY-1215 treatment is mediated through increased CHOP expression. Taken together, our findings indicate that oncogenic BRAF induces chronic ER stress and that inducers of acute ER stress could be a novel treatment strategy for poor prognostic BRAFMT colorectal cancer. Mol Cancer Ther; 17(6); 1280-90. ©2018 AACR.

P-355 - Combination of proteasome inhibitors with temozolomide to increase the anti-tumor effect in 3D culture model of glioblastoma

Proteasomal degradation is an important target in cancer therapy. Many inhibitors have been developed and bortezomib and carfilzomib are used in the clinics. Glioblastoma is a grade IV astrocytoma, and the treatment procedure is radiotherapy and temozolomide combination following surgery. But many cases show drug resistance and recurrence. Recently glioblastoma stem cells, with high protesome activity, were found to be responsible for the drug resistance. In this study, we developed a 3D culture model of glioblastoma and we tested bortezomib and carfilzomib in combination with temozolomide. Hybrid 3D nanofiber scaffold were prepared by electrospinning of core-shell poly(e-caprolactone) (PCL)- bacterial cellulose and PCL- hyaluronic acid. Morphological, surface, and mechanical structure of 3D scaffold were characterized. Attachment of glioblastoma on the scaffold was confirmed by electron microscopy. Temozolomide, bortezomib and carfilzomib treatments were performed on the cell attached scaffolds. Proteasome inhibition, cell viability and apoptosis were tested. Data showed that combination of bortezomib and carfilzomib with temozolomide increased the anti-tumor effects. According to our previous studies, carfilzomib with its lower side effects can be tried in the clinics.

Loss of C/EBP-β LIP drives cisplatin resistance in malignant pleural mesothelioma

ObjectivesCisplatin-based chemotherapy is moderately active in malignant pleural mesothelioma (MPM) due to intrinsic drug resistance and to low immunogenicity of MPM cells. CAAT/enhancer binding protein (C/EBP)-β LIP is a pro-apoptotic and chemosensitizing transcription factor activated in response to endoplasmic reticulum (ER) stress. Materials and methodsWe investigated if LIP levels can predict the clinical response to cisplatin and survival of MPM patients receiving cisplatin-based chemotherapy. We studied the LIP-dependent mechanisms determining cisplatin-resistance and we identified pharmacological approaches targeting LIP, able to restore cisplatin sensitiveness, in patient-derived MPM cells and animal models. Results were analyzed by a one-way analysis of variance test. ResultsWe found that LIP was degraded by constitutive ubiquitination in primary MPM cells derived from patients poorly responsive to cisplatin. LIP ubiquitination was directly correlated with cisplatin chemosensitivity and was associated with patients’ survival after chemotherapy. Overexpression of LIP restored cisplatin’s pro-apoptotic effect by activating CHOP/TRB3/caspase 3 axis and up-regulating calreticulin, that triggered MPM cell phagocytosis by dendritic cells and expanded autologous anti-tumor CD8+CD107+T-cytotoxic lymphocytes.Proteasome inhibitor carfilzomib and lysosome inhibitor chloroquine prevented LIP degradation. The triple combination of carfilzomib, chloroquine and cisplatin increased ER stress-triggered apoptosis and immunogenic cell death in patients’ samples, and reduced tumor growth in cisplatin-resistant MPM preclinical models. ConclusionThe loss of LIP mediates cisplatin resistance, rendering LIP a possible predictor of cisplatin response in MPM patients. The association of proteasome and lysosome inhibitors reverses cisplatin resistance by restoring LIP levels and may represent a new adjuvant strategy in MPM treatment.

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.

Protein Homeostasis: A Key Target in Myeloma

Multiple myeloma is a disease of long-lived plasma cells that remains incurable in the majority of patients. However advances in the treatment have been made in the last 20 years resulting in significant improvement in the survival of patients with this disease. Interestingly almost all of these advances have been made through the targeting of pathways or molecules that are shared between normal and malignant plasma cells and are not due to the precision targeting of myeloma cell dependencies associated with the transformation process. Most notably targeting both specific and bulk protein homeostasis through the action of IMiDs and proteasome inhibitors have become the backbone for the treatment of newly diagnosed myeloma in the United States. We have focused on proteasome function and regulation to better understand why a macromolecular structure that is present and functions in every cell in the body is such an effective target for myeloma therapy. Our initial findings indicated that proteasome inhibition activated an endoplasmic reticulum stress response known as the unfolded protein response (UPR). The UPR is required for normal differentiation and maintenance of plasma cells, although our initial findings suggesting that the PERK arm of the response, which is not required for normal plasma cell maintenance was activated by proteasome inhibitors. We determined that the likely source of unfolded protein was immunoglobulin produced by myeloma cells and that the fate of immunoglobulin was a key determinant of sensitivity to proteasome inhibition. We have now focused on additional factors that determine the response to proteasome inhibition and consistent with previous findings have demonstrated that activation of heat shock response is a early protective event following proteasome inhibition. However we were unable to demonstrate that any single heat shock protein was sufficient to alter the response to proteasome inhibition. Rather it was the activation of the heat shock response itself and that protection was only efficiently blocked by inhibition of the transcriptional regulator of the response, HSF1. HSF1 is activated through post-translational modifications and proteasome inhibition results in phosphorylation of HSF1 at several sites, most notably S326. This can be blocked with the multi-kinase inhibitor TG02 that effectively blocks the proteasome inhibitor-induced heat shock response and sensitizes myeloma cells to proteasome inhibitor-induced cell death. Based on these findings, the combination of TG02 and carfilzomib are being tested clinically. The cellular response to proteasome inhibitors is also controlled through the regulation of proteasome assembly. We have recently demonstrated that 14-3-3ζ an anti-apoptotic phospho-serine binding protein, can limit proteasome assembly and activity through its binding to the PA28α subunit of the 11S regulator. This significantly lowers proteasome capacity and sensitizes cells to proteasome inhibition but not to other therapeutic agents. Finally in addition to regulation of bulk protein homeostasis, targeting of specific protein turnover has proven effective in myeloma treatment. IMiDs can alter the ability of the E3 ligase cereblon to recognize substrates for ubiquitination, resulting in the degradation of key B cell transcription factors IKZF1/3. These can regulate the plasma cell maintenance factor IRF4 as well as play a direct role in the regulation plasma cell superenhancers that control the expression of oncogenes translocated in myeloma. Another key mediator of myeloma cell survival, MCL1 is regulated via protein stability and can targeted in this manner. Direct and indirect targeting of MCL1 will be discussed. Disclosures Boise: Abbvie: Consultancy; Eli Lilly and Company: Research Funding.

A Phase Ib/IIa Trial of the Combination of Romidepsin, Lenalidomide and Carfilzomib in Patients with Relapsed/Refractory Lymphoma Shows Complete Responses in Relapsed and Refractory B- and T-Cell Lymphomas

A Phase Ib/IIa Trial of the Combination of Romidepsin, Lenalidomide and Carfilzomib in Patients with Relapsed/Refractory Lymphoma Shows Complete Responses in Relapsed and Refractory B- and T-Cell Lymphomas