Differential Activity of Pazopanib and Imatinib Against PDGFRA-Related MPN and AML

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5153-5153
Author(s):  
Steffen Koschmieder ◽  
Mirle Schemionek ◽  
Christian Elling ◽  
Utz Krug ◽  
Torsten Kessler ◽  
...  
Keyword(s):  
Cell Growth ◽  
Tyrosine Kinase Inhibitor ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Kinase Inhibitor ◽  
Point Mutations ◽  
Differential Sensitivity ◽  
Advisory Committees

Abstract Abstract 5153 Introduction: Pazopanib is a tyrosine kinase inhibitor with proven activity against metastatic renal cancer. Due to its target spectrum of kinases (PDGFR, KIT, and VEGFR), we sought to investigate its activity against myeloid malignancies. Methods: 32D cells transduced with FIP1L1-PDGFRA or several activating PDGFRA point mutations as well as AML cell lines were analyzed for the effects of pazopanib vs. imatinib on cell growth, MTS activity, 7-AAD positivity, and clonogenic growth. Pazopanib and imatinib were purchased from LC Laboratories Woburn, MA, USA. Results: Pazopanib was found to decrease the growth of FIP1L1-PDGFRA transduced cell lines at low nanomolar concentrations (10 nM). 1000 nM doses were equally effective as 1000 nM of the PDGFR tyrosine kinase inhibitor imatinib. MTS assays confirmed that at 10 nM, pazopanib reduced cell proliferation to 28% of that of vehicle-treated control cells (DMSO 0.05%), while 100 nM of pazopanib completely abolished cell growth and suppressed MTS activity. Analysis of 7-AAD positivity using flow cytometry showed that reduction in cell growth and MTS activity was due to induction of apoptosis (17+/−0.6%, 63+/−0.5%, and 87+/−0.5%, 86+/−0.1% for 10, 100, and 1000 nM of pazopanib and 1000 nM of imatinib, respectively). Interestingly, while two PDGFRA activating point mutations (H650Q and R748G) recently identified in patients with idiopathic hypereosinophilic syndrome-type myeloproliferative neoplasms (MPN) were equally sensitive against pazopanib and imatinib, two other such point mutations showed differential sensitivity, with Y849S being more sensitive to imatinib and N659S being more sensitive to pazopanib. When evaluating the effect of pazopanib on acute myeloid leukemia cell lines, we found that imatinib inhibited the cell growth and reduced colony forming units of Kasumi-1 and BCR-ABL positive K562 but not MV4-11 or NB-4 cells, while Kasumi-1, MV4-11, and NB-4 cells were sensitive towards pazopanib treatment. Moreover, while the clonogenic growth of bone marrow-derived cells from two control patients (lymphoma or AML in remission) were only reduced to 75% of control with pazopanib treatment in vitro (100 nM), 100 nM pazopanib decreased colony growth to 18% in another patient with AML at diagnosis. Conclusion: Our data suggest that pazopanib may be active against a variety of myeloid neoplasms and that clinical studies assessing its efficacy are warranted. Also, cells may show differential sensitivity against the PDGFR and KIT inhibitors pazopanib and imatinib. Disclosures: Koschmieder: GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Off Label Use: Data on in vitro activity of pazopanib and imatinib in MPN and AML discussed. Krug:MedA Pharma: Honoraria; Novartis: Honoraria; Alexion: Honoraria; Boehringer Ingelheim: Research Funding; Sunesis: Honoraria. Müller-Tidow:Novartis: Research Funding.

ATP Dependent Efflux Transporters ABCB1 and ABCG2 Are Unlikely to Impact the Efficacy, or Mediate Resistance to the Tyrosine Kinase Inhibitor, Ponatinib

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2745-2745 ◽  
Author(s):  
Deborah L. White ◽  
Liu Lu ◽  
Timothy P. Clackson ◽  
Verity A Saunders ◽  
Timothy P Hughes
Keyword(s):  
Tyrosine Kinase ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Kinase Inhibitor ◽  
K562 Cells ◽  
Temperature Dependent ◽  
Advisory Committees ◽  
Significant Difference

Abstract Abstract 2745 Ponatinib is a potent pan-BCR-ABL tyrosine kinase inhibitor (TKI) currently in a pivotal phase 2 clinical trial. Ponatinib (PON) was specifically designed to target both native and all mutant forms of BCR-ABL, including T315I. The phase I study of oral ponatinib in patients with refractory CML/ALL or other hematologic malignancies recently reported that 66% and 53% of patients with CP-CML achieved MCyR and CCyR respectively (Cortes et al., ASH 2011 abstract #210). While extensive modelling experiments in BaF3 cells have been performed characterising in vitro response to ponatinib, little is known about the interactions of this drug and drug transporters that impact the response of other tyrosine kinase inhibitors (TKIs). To explore this we have examined both the degree of in vitro kinase inhibition mediated by ponatinib in BCR-ABL+ cell lines, and the intracellular uptake and retention (IUR) of ponatinib achieved. The IC50 was determined by assessing the reduction in %p-Crkl in response to increasing concentrations of ponatinib in vitro. The IUR assay was performed as previously using [14-C]-ponatinib. To determine the role of ABCB1 and ABCG2, both previously implicated in the transport of other TKIs, IC50 analysis was performed on K562 cells, and variants; ABCB1 overexpressing K562-DOX and ABCG2 overexpressing K562-ABCG2. As shown in Table 1, in contrast to the results previously observed with imatinib (IM), nilotinib (NIL) and dasatinib (DAS) there was no significant difference in the IC50ponatinib between these three cell lines, suggesting neither ABCB1 nor ABCG2 play a major role in ponatinib transport. Furthermore, the addition of either the ABCB1 and ABCG2 inhibitor pantoprazole, or the multidrug resistance (MDR) inhibitor cyclosporin did not result in a significant change in the IC50ponatinib in any of the cell lines tested. In contrast the addition of either pantoprazole or cyclosporin resulted in a significant reduction in IC50IM, IC50NIL. and IC50DAS of K562-DOX cells, supporting the notion that these TKIs interact with ABCB1.Table 1:The IC50 of ponatinib (compared to IM, NIL and DAS) in K562 cells and the over-expressing variants DOX and ABCG2 in the presence of the ABC inhibitors pantoprazole and cyclosporin. n=5. *p<0.05IC50% reduction in IC50+ pantoprazole+ cyclosporinPON (nM)IM (μM)NIL (nM)DAS (nM)PONIMNILDASPONIMNILDASK5627.793751111544*NA−107NA2DOX7.919*598*100*1018*63*1655*88*ABCG26.4730025*6NA To further examine the effect of ABC transporters on ponatinib efflux we have determined the IUR of [14-C]-ponatinib in K562, DOX and ABCG2 cell lines. We demonstrate no significant difference in the IUR between these cell lines at 37°C (n=6) (K562 vs DOX p=0.6; K562 vs ABCG2 p=0.37 and DOX vs ABCG2 p=0.667 at 2uM respectively). Temperature dependent IUR experiments reveal a significant reduction in the ponatinib IUR at 4°C compared to 37°C in K562 cells (n=6) (p=0.008), DOX cells (p=0.004) and ABCG2 cells (p=0.002) supporting the likely involvement of an ATP/temperature dependent, and yet to be determined, component of ponatinib influx. There was no significant difference in the IUR between these cell lines at 4°C (p=0.824, p=0.7 and p=0.803 respectively). Importantly, these data are consistent with the IC50ponatinib findings. If ATP dependent efflux pumps (ABCB1 and ABCG2) were actively transporting ponatinib, a significant decrease in IUR in DOX and ABCG2 at 37°C compared to K562 cells would be expected, but is not observed here. Analysis of ponatinib IUR in the prototypic ABCB1 over-expressing CEM-VBL100 cells, and their parental, ABCB1 null counterparts (CCRF-CEM) further confirmed these findings. The IUR in VBL100 cells was significantly higher than that observed in CEM's (p<0.001; n=5), providing further evidence that ponatinib was not being exported from the cell actively via ABCB1. These data suggest that the transport of ponatinib is, at least in part, temperature-dependent indicating a yet to be determined ATP transporter may be involved in the transport of ponatinib into leukaemic cells. Importantly, this data suggests that ponatinib is unlikely to be susceptible to resistance via the major ATP efflux transporters (ABCB1 or ABCG2) that have been previously demonstrated to significantly impact the transport of, and mediate resistance to other clinically available TKIs. Disclosures: White: BMS: Honoraria, Research Funding; Novartis Pharmaceuticals: Honoraria, Research Funding. Clackson:ARIAD: Employment. Hughes:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; ARIAD: Honoraria, Membership on an entity's Board of Directors or advisory committees.

POU2AF1 As a Master Regulator of Oncogenic Transcription Factor Networks in Myeloma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 18-19
Author(s):  
Ricardo De Matos Simoes ◽  
Ryosuke Shirasaki ◽  
Huihui Tang ◽  
Shizuka Yamano ◽  
Benjamin G Barwick ◽  
...  
Keyword(s):  
Cell Growth ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Rna Seq ◽  
Advisory Committees ◽  
Genome Scale ◽  
Crispr Activation

Background: Functional genomics studies based on CRISPR and shRNA have documented that multiple myeloma (MM) cells are preferentially dependent (compared to other neoplasias) on a series of TFs, including IKZF1 and IKZF3 (which are targeted by thalidomide derivatives) and others that are not amenable to degradation or small molecule inhibition. Transcriptional co-factors have been therapeutically targeted, for example, inhibitors of BRD4, a co-factor for pTEFB, can be used to down-regulate c-myc. Aim: To identify new transcriptional vulnerabilities in MM with an emphasis on transcriptional co-factors Methods: We integrated results from genome-scale studies using the AVANA library for loss-of-function by gene editing (in 19 MM lines) and the Calabrese library for CRISPR-mediated gene activation (in 5 MM lines) to identify critical transcriptional co-factors (co-TFs). RNA-Seq analysis was used to identify critical pathways affected by POU2AF1 activation and existing ChIP-Seq tracks in MM cells were reanalyzed. Results: POU2AF1 (OCA-B) was the most preferentially essential TF co-factor in MM cell lines vs. non-MM and one of top genes which, upon CRISPR activation in genome-scale studies, increased MM cell fitness in vitro. We further confirmed the role of this gene using focused libraries of sgRNAs against POU2AF1 in vitro and in an in vivo model of MM cell growth in bone marrow-like scaffolds "functionalized" with humanized mesenchymal bone marrow stromal cells to simulate the human BM. CRISPR activation of POU2AF1 is associated with increased MM cell growth. RNA-Seq of POU2AF1 activation in LP1 cells a transcriptional program characterized by upregulation of other genes that are preferentially essential for MM including PRDM1, SUPT7L, UBE2G2 and TSC1; broad-spectrum oncogenic dependencies (e.g KRAS) and genes known or proposed to be involved in the pathophysiology of MM or other neoplasias (e.g. RUNX2, FGFR3, SMO, CREB5, TNFRSF13B, MEF2D, PCGF2). POU2AF1 overexpression was also associated with down-regulation of CDKN1C; of MHC class II molecules and their transcriptional activator CIITA, suggesting that POU2AF1 activation could also contribute to increased MM growth in vivo by allowing escape from immune surveillance. ATAC-Seq data and genome-wide ChIPseq for H3K27Ac in MM cell lines indicate that chromatin surrounding the POU2AF1 locus was highly accessible, concordant with the consistent expression of this TF in MM cell lines and patient-derived cells. CoMMpass data showed that POU2AF1 expression was enhanced in a subset of MM patients at relapse compared to diagnosis. Motif analysis of ChIP-seq data for POU2AF1 identified significant overlap with motifs for TFs relevant to the POU family (e.g. Oct11, Oct2, Oct4); members of the ETS family (e.g. ELF1, Elf4, GABPA); and other TFs with roles in MM including c-myc; IRF4; NF-kappaB, PRDM1, RUNX2 and the POU2AF1 target CREB5. These data suggest a functional interaction between POU2AF1 and other MM-relevant TFs. The transcriptional signature of POU2AF1 activation is enriched for genes downregulated by suppression/inhibition of MM-preferential TFs or epigenetic regulators including IRF4, PRDM1, IKZF1/3 and DOT1L. POU2AF1 binding motifs are also enriched in the promoter regions of MM-preferential dependencies including several MM-preferential TFs. Conclusions: POU2AF1 is essential for MM cells in vitro and in vivo; has a significantly more pronounced and recurrent role as a dependency in MM compared to most other neoplasias; and can further drive MM cell growth, through its ability to interact with several TFs critical for MM, forming multi-protein functional complexes. These results establish POU2AF1 as a central component in the regulatory network of oncogenic TFs in MM and highlight the value of further exploring POU2AF1 as a therapeutic target in MM. Disclosures Downey-Kopyscinski: Rancho BioSciences, LLC: Current Employment. Tsherniak:Cedilla Therapeutics: Consultancy; Tango Therapeutics: Consultancy. Boise:AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genetech: Membership on an entity's Board of Directors or advisory committees. Mitsiades:FIMECS: Consultancy, Honoraria; Ionis Pharmaceuticals, Inc.: Consultancy, Honoraria; Arch Oncology: Research Funding; Janssen/Johnson & Johnson: Research Funding; Karyopharm: Research Funding; TEVA: Research Funding; Takeda: Other: employment of a relative; Fate Therapeutics: Consultancy, Honoraria; Sanofi: Research Funding; Abbvie: Research Funding; EMD Serono: Research Funding.

A Novel Acanthoic Acid Analog NPI-1342 Blocks IκB Kinase-α and Trigger In Vitro and In Vivo cytotoxicity in Multiple Myeloma Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1841-1841
Author(s):  
Dharminder Chauhan ◽  
Ajita V. Singh ◽  
Arghya Ray ◽  
Teru Hideshima ◽  
Paul G. Richardson ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
Board Of Directors ◽  
Tumor Cells ◽  
Cell Lines ◽  
Inhibitory Effect ◽  
Advisory Committees ◽  
Acid Analog

Abstract Abstract 1841 Introduction: The dimeric Nuclear Factor-kappa B (NF-κB) transcription factor plays a key role during multiple myeloma (MM) cell adhesion-induced cytokine secretion in bone marrow stromal cells, which in turn triggers MM cell growth in a paracrine manner. NF-κB signaling pathway is mediated via canonical (IKK-α/IKK-β/NEMO-P50/65 or NF-κB1) and non-canonical (IKK-α/IKK-α/NIK-p52/RelB or NF-κB2) components. Prior studies have also linked constitutive activation of non-canonical NF-κB pathway to genetic abnormalities/mutation, allowing for an autocrine growth of MM cells. Other recent studies showed that constitutive NF-κB activity in tumor cells from MM patients renders these cells refractory to inhibition by bortezomib; and in fact, that bortezomib induces canonical NF-κB activity. These reports provided the impetus for the development of an agent with ability to modulate canonical and/or non-canonical NF-κB axis, allowing for a more robust and specific inhibition of NF-κB. Recent research and development efforts at Nereus Pharmaceuticals, Inc., have identified a novel small molecule acanthoic acid analog NPI-1342 as a potent NF-κB inhibitor. Here, we examined the effects of NPI-1342 on canonical versus non-canonical NF-κB signaling pathways, as well as its anti-tumor activity against MM cells using both in vitro and in vivo model systems. Methods: We utilized MM.1S, MM.1R, RPMI-8226, U266, KMS12PE, NCI-H929, OCI-MY5, LR5, Dox-40, OPM1, and OPM2 human MM cell lines, as well as purified tumor cells from patients with MM. Cell viability assays were performed using MTT and Trypan blue exclusion assays. Signal transduction pathways were evaluated using immunoblot analysis, ELISA, and enzymology assays. Animal model studies were performed using the SCID-hu model, which recapitulates the human BM milieu in vivo. Results: We first examined the effects of NPI-1342 on lipopolysaccharides (LPS)-induced NF-κB activity. Results showed that NPI-1342 inhibits LPS-stimulated NF-κB activity in vitro, as measured by phosphorylation of IkBa. To determine whether NPI-1342 triggers a differential inhibitory effect on IKKβ versus IKKα, MM.1S MM cells were treated with NPI-1342 for 48 hours, and protein lysates were subjected to kinase activity assays. NPI-1342 blocked IKKα, but not IKKβ or IKKγ phosphorylation. We next assessed whether the inhibitory effect of NPI-1342 on NF-κB activity is associated with cytotoxicity in MM cells. We utilized a panel of MM cell lines: at least five of these have mutations of TRAF3 (MM.1S, MM.1R, DOX40 and U266); one has no known NF-κB mutations (OPM2), and one has amplification of NF-κB1 (OCI-MY5). Treatment of MM cell lines and primary patient (CD138 positive) MM cells for 48 hours significantly decreased their viability (IC50 range 15–20 μM) (P < 0.001; n=3) without affecting the viability of normal peripheral blood mononuclear cells, suggesting selective anti-MM activity and a favorable therapeutic index for NPI-1342. NPI-1342-induced a marked increase in Annexin V+ and PI- apoptotic cell population (P < 0.001, n=3). Mechanistic studies showed that NPI-1342-triggered apoptosis in MM cells is associated with activation of caspase-8, caspase-9, caspase-3, and PARP cleavage. We next examined the in vivo effects of NPI-1342 in human MM xenograft models. For these studies, we utilized the SCID-hu MM model, which recapitulates the human BM milieu in vivo. In this model, MM cells are injected directly into human bone chips implanted subcutaneously in SCID mice, and MM cell growth is assessed by serial measurements of circulating levels of soluble human IL-6R in mouse serum. Treatment of tumor-bearing mice with NPI-1342 (20 mg/kg intraperitoneally, QD1-5 for 2 weeks), but not vehicle alone, significantly inhibits MM tumor growth in these mice (10 mice each group; P = 0.004). The doses of NPI-1342 were well tolerated by the mice, without significant weight loss. Finally, immunostaining of implanted human bone showed robust apoptosis and blockade of NF-κB in mice treated with NPI-1342 versus vehicle alone. Conclusions: We demonstrate the efficacy of a novel small molecule inhibitor of NF-κB NPI-1342 in MM using both in vitro and in vivo models. NPI-1342 blocks NF-κB activity with a preferential inhibitory activity against IKK-α component of NF-κB signaling. Our preclinical studies support evaluation of NPI-1342 as a potential MM therapy. Disclosures: Hideshima: Acetylon: Consultancy. Richardson:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Palladino:Nereus Pharmaceuticals, Inc: Employment, Equity Ownership. Anderson:Celgene: Consultancy; Millennium: Consultancy; Onyx: Consultancy; Merck: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Acetylon:; Nereus Pharmaceuticals, Inc: Consultancy.

Increasing Expression Of The Efflux Transporter ABCB1 May Predispose CML Cells To Overt TKI Resistance

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5157-5157
Author(s):  
Laura Eadie ◽  
Timothy P. Hughes ◽  
Deborah L. White
Keyword(s):  
Disease Progression ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Resistance Mechanisms ◽  
Molecular Response ◽  
Advisory Committees ◽  
Tki Resistance ◽  
Additional Resistance

Abstract Tyrosine kinase inhibitors (TKIs) result in excellent responses in most Chronic Myeloid Leukemia (CML) patients. However, up to 35% of patients treated with imatinib (IM) exhibit resistance and more recently nilotinib (NIL) and dasatinib (DAS) resistance have also been observed. Mutations in the BCR-ABL kinase domain (KD) are the main cause of secondary TKI resistance. Other mechanisms include overexpression of BCR-ABL, LYN and ABCB1. Predicting patients with susceptibility to mutation development and disease progression is crucial, thus we investigated the kinetics of TKI resistance emergence in vitro and in vivo. ABCB1 is implicated in TKI efflux hence we postulated that overexpression of ABCB1 leads to reduced intracellular TKI concentrations, resulting in inferior inhibition of Bcr-Abl predisposing cells to resistance development. Accordingly, 3 CML blast crisis (BC) cell lines (K562, K562-Dox, KU812) were cultured in increasing concentrations of IM to 2 μM, NIL to 2 μM and DAS to 200 nM until we observed overt resistance defined as a significant increase in survival in cytotoxicity assays and p-Crkl dependent IC50. Mechanisms of resistance were investigated in cell line intermediates: BCR-ABL, ABCB1 and LYN mRNA expression levels were determined by RT-PCR and KD mutation sequencing was performed. In our TKI resistant cell lines (Table 1), an increase in ABCB1 mRNA was the initial change observed prior to the development of additional resistance mechanisms (KD mutations, ABCB1 BCR-ABL and LYN overexpression). Interestingly, in 4/6 cells lines ABCB1 mRNA reduced to basal levels or below following establishment of these additional resistance mechanisms. ABCB1 levels were assessed in 37 de novo CML patients treated with IM who achieved major molecular response (MMR) compared with patients who progressed to BC, lost MMR or developed KD mutations. ABCB1 levels were determined in blood at diagnosis and following therapy (selected patients summarized in Table 2). A sustained >2 fold rise in ABCB1 was observed prior to disease progression in 3/3 patients and in 13/16 patients who did not achieve MMR. Importantly, the same was not observed in patients who achieved MMR (1/6 patients). The fold change of ABCB1 mRNA at day 22 vs diagnosis in patients achieving MMR was significantly different to that in patients not achieving MMR (p=0.004). ABCB1 increased by >2 fold post therapy and decreased following mutation development in 3/12 patients, confirming observations made in vitro, while 6/12 patients demonstrated sustained increase in ABCB1 post mutation similar to results observed in progression patients. ABCB1 mRNA did not change during therapy in 3/12 patients with mutations. While we recognize the majority of cells present in patients who achieve MMR are normal rather than leukemic, it is important to note that in patients who do not achieve MMR, ABCB1 expression increases in the remaining leukemic cells. We conclude ABCB1 overexpression acts as an initial mediator of resistance, providing a favorable environment for development of further resistance. Sustained increased levels of ABCB1 may contribute to disease progression and lack of response to IM. Additionally, ABCB1 may serve as a prognostic indicator (eg: level at day 22) and potentially assist in development of treatment strategies using TKIs in combination with other medications to enhance intracellular TKI concentration. Disclosures: Hughes: Ariad: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; CSL: Research Funding. White:Novartis: Research Funding; BMS: Research Funding, Speakers Bureau; Ariad: Research Funding; CSL: Research Funding.

scholarly journals The 3-Drug Combination Treatment ART838, ABT199 Plus Sorafenib Is Effective Against AML Xenografts, Possibly Via Cooperative Targeting of MCL1

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4044-4044
Author(s):  
Blake S Moses ◽  
Jennifer Fox ◽  
Xiaochun Chen ◽  
Samantha McCullough ◽  
Sang Ngoc Tran ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Board Of Directors ◽  
Cell Lines ◽  
Drug Combination ◽  
Research Funding ◽  
Leukemia Cell ◽  
Advisory Committees ◽  
Leukemia Cell Lines ◽  
Equity Ownership

Abstract Antimalarial artemisinins have broad antineoplastic activity in vitro, are well tolerated and inexpensive, and can be parenterally or orally administered in humans. Artemisinin-derived trioxane diphenylphosphate dimer 838 (ART838; a potent artemisinin-derivative) inhibited acute leukemia growth in vivo and in vitro, at doses where normal human CD34+ hematopoietic stem-progenitor cell clonogenicity was essentially unaffected (Fox et al, Oncotarget 2016, PMID: 26771236). In our focused drug combination screen for drugs that synergize with ART838, the only BCL2 inhibitors in the screen library of 111 emerging antineoplastic compounds, navitoclax (ABT737) and venetoclax (ABT199; FDA-approved), were identified as 2 of the top 3 candidates. Synergies between ART838 and BCL2 inhibitors were validated in multiple acute leukemia cell lines and primary cases. This ART838-BCL2 inhibitor synergy may be due to reduced levels of MCL1 protein that we and others have observed in multiple acute leukemia cell lines and primary cases treated with artemisinins (Budhraja et al, Clin Cancer Res 2017, PMID: 28974549). Treatment of acute leukemia xenografts with the ART838 plus ABT199 combination reduced leukemia growth rates and prolonged survivals, compared to vehicle or either ART838 or ABT199 alone. To add to the efficacy of this ART838 plus ABT199 treatment regimen, we sought to rationally add a third low-toxicity active antileukemic agent. Sorafenib (SOR; FDA-approved) inhibits multiple kinases which may mediate its antileukemic activity, with the importance of the targets varying from case to case; e.g. FLT3 is an important target in many AMLs. In addition, several reports have found that SOR reduces MCL1 protein stability and translation through inhibition of the ERK and PI3K pathways (Wang et al, Clin Cancer Res 2016, PMID: 26459180; Huber et al, Leukemia 2011, PMID: 21293487). In all acute leukemia cell lines tested, we observed large reductions in MCL1 protein levels with SOR treatment, which may further rationalize the addition of SOR to our ART838 plus ABT199 antileukemic regimen. We had previously observed strong in vitro synergy between ART838 and SOR (PMID: 26771236). Treatment of acute leukemia xenografts with the ART838 plus SOR combination reduced leukemia xenograft growth rates and prolonged survivals, compared to single drugs. Mice bearing luciferase-labelled acute leukemia xenografts were treated (PO daily x5) with single drug or 2-drug or 3-drug combinations of ART838, ABT199, and SOR, each at their individual maximally tolerated doses. Treatment with this 3-drug combination caused rapid regression of luciferase-labelled MV4;11 AML xenografts (Fig 1A). The 5-day treatment cycles were repeated every other week, and mice receiving this 3-drug combination survived >4 times longer than vehicle-treated mice (Fig 1B). Mouse body weights were stable during treatment. Although myelosuppression is the human clinical dose-limiting toxicity of each of these 3 drugs, mouse blood cell counts during 3-drug combination treatment were in the normal range. Treatment of a luciferase-labelled primary AML leukemia xenograft with this 3-drug combination reduced leukemia growth more than the single drugs or 2-drug combinations (Fig 1C). Assessment of efficacy and pharmacokinetics-pharmacodynamics against diverse acute leukemia xenografts will test this combination of ART838, ABT199 plus SOR as a rational low-toxicity drug triad for treatment of acute leukemias and potentially other cancers. Disclosures Fox: Intrexon Corporation: Employment. Tyner:Genentech: Research Funding; Janssen: Research Funding; AstraZeneca: Research Funding; Gilead: Research Funding; Incyte: Research Funding; Constellation: Research Funding; Array: Research Funding; Takeda: Research Funding; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Aptose: Research Funding. Civin:ConverGene LLC: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; GPB Scientific LLC: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; 3DBioWorks Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; BD (Becton Dickinson): Honoraria.

scholarly journals For Patients with Sustained MR4-MR4.5, Less Frequent Molecular Monitoring during the First 12 Months after Tyrosine Kinase Inhibitor Cessation Is Viable for Timely Detection of Loss of MMR

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1890-1890
Author(s):  
Naranie Shanmuganathan ◽  
Susan Branford ◽  
Jodi Braley ◽  
Devendra Hiwase ◽  
David T. Yeung ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitor ◽  
Board Of Directors ◽  
Median Time ◽  
Research Funding ◽  
Kinase Inhibitor ◽  
Molecular Response ◽  
Monitoring Strategy ◽  
Molecular Monitoring ◽  
Advisory Committees

Abstract Background: Discontinuation of tyrosine kinase inhibitor (TKI) treatment for chronic myeloid leukaemia (CML) patients in stable deep molecular response leads to treatment-free remission (TFR) in approximately 50% of cases. In most studies, monthly PCRs was performed for 12 months followed by 2-3 monthly testing thereafter. Around 80% of molecular relapses occur within the first 6 months after TKI cessation. The current recommendation for TKI recommencement is a single BCR-ABL1 value ≥0.1% IS (International scale), indicating loss of major molecular response (MMR). Not all institutions can offer monthly PCR monitoring due to financial constraints, particularly relevant in developing countries. For some patients, remaining on TKI is a cheaper alternative. Aim: To assess the safety of less frequent BCR-ABL1 monitoring for detection of loss of MMR for CML patients attempting TFR. Methods: We monitored 85 patients who ceased TKI with the aim of achieving TFR. Patients had a minimum of 24 months of sustained MR4 (n=3) or MR4.5 (n=82) prior to TKI cessation. At the time of TKI cessation, 64 patients were on imatinib (75%), 17 on nilotinib (20%) and 4 on dasatinib (5%). Forty of the patients were enrolled in the TWISTER study where the criteria for TKI recommencement was loss of MMR or 2 consecutively rising BCR-ABL1 positive values. The remaining patients were on a registry study and the trigger for TKI recommencement was loss of MMR. Results: TKI recommencement occurred in 49 of 85 patients. Median time to TKI recommencement was 4 months (range 2-28 months) at a median BCR-ABL1 value of 0.27% on the International Scale (IS), range 0.002-24% IS. Thirty-six of the 49 patients (73%) lost MMR prior to TKI recommencement; the median time to loss of MMR was 3 months (range 1 to 10 months). One patient lost MMR within the first month. Figure A demonstrates the time to loss of MMR in the 36 patients with PCR values ≥ 0.1%. Eighteen of the 36 patients (50%) lost MMR by the 3 month BCR-ABL1 assessment and 35 of 36 patients (97%) lost MMR by 6 months. The latest loss of MMR was at 10 months. Fourteen patients recommenced TKI at a BCR-ABL1 value of >1% and 1 recommenced at a value >10%. Clinician delay in TKI recommencement of 1 month resulted in a BCR-ABL1 rise from 0.84% to 24% with associated loss of complete hematological response. We propose monthly BCR-ABL1 testing between 2 and 6 months post TKI cessation followed by 2 monthly testing. Detection of a BCR-ABL1 value of ≥0.1% would trigger TKI recommencement. In the presence of a rising BCR-ABL1, which remains ≤0.1%, monthly monitoring should ensue in order to avoid hematological relapse. If this strategy were employed in this cohort of patients, only 1 patient would have had the trigger for TKI recommencement delayed by 1 month (estimated BCR-ABL1 at recommencement ~2.5%). This patient had loss of MMR in the first month post TKI cessation. If this molecular monitoring strategy was applied to patients in our cohort who had not lost MMR at TKI recommencement, we estimate that 1 other patient would have had TKI recommencement delayed by 1 month based on the average BCR-ABL1 doubling time of 1 log per month. A proportion of patients maintain low levels of BCR-ABL1 after TKI cessation and do not lose MMR. There were 2 such patients in our cohort. Conclusion: The critical time for molecular monitoring to trigger TKI recommencement is the first 6 months. A monthly monitoring strategy beginning 2 months after cessation would capture the majority of patients at loss of MMR. The data suggest that after 6 months, 2-monthly monitoring could follow. Monthly BCR-ABL1 testing can be re-introduced in the event of a positive result in those that ceased TKI with undetectable BCR-ABL1 or if there is a BCR-ABL1 result higher than the cessation value. This approach would reduce BCR-ABL1 testing by approximately 33% in the majority of cases while minimizing hematological relapse. Therefore this strategy would reduce the cost and inconvenience of molecular monitoring for a trial of TKI cessation, making the option of TFR available to some patients for whom it is otherwise not feasible. Disclosures Branford: Qiagen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Cepheid: Consultancy; Ariad: Research Funding; Bristol Myers Squibb: Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Yeung:BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Research Funding. Ross:Novartis Pharmaceuticals: Honoraria, Research Funding; BMS: Honoraria. Hughes:Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Australasian Leukaemia and Lymphoma Group (ALLG): Other: Chair of the CML/MPN Disease Group.

scholarly journals Targeting Wnt/β-Catenin and BCL-2, Two Critical Survival Factors, Synergistically Induces Apoptosis in AML Via Rac1-Mediated MCL-1 Inhibition

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1442-1442
Author(s):  
Xiangmeng Wang ◽  
Po Yee Mak ◽  
Wencai Ma ◽  
Xiaoping Su ◽  
Hong Mu ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Single Agent ◽  
Advisory Committees ◽  
Equity Ownership ◽  
Critical Survival

Abstract Wnt/β-catenin signaling regulates self-renewal and proliferation of AML cells and is critical in AML initiation and progression. Overexpression of β-catenin is associated with poor prognosis. We previously reported that inhibition of Wnt/β-catenin signaling by C-82, a selective inhibitor of β-catenin/CBP, exerts anti-leukemia activity and synergistically potentiates FLT3 inhibitors in FLT3-mutated AML cells and stem/progenitor cells in vitro and in vivo (Jiang X et al., Clin Cancer Res, 2018, 24:2417). BCL-2 is a critical survival factor for AML cells and stem/progenitor cells and ABT-199 (Venetoclax), a selective BCL-2 inhibitor, has shown clinical activity in various hematological malignancies. However, when used alone, its efficacy in AML is limited. We and others have reported that ABT-199 can induce drug resistance by upregulating MCL-1, another key survival protein for AML stem/progenitor cells (Pan R et al., Cancer Cell 2017, 32:748; Lin KH et al, Sci Rep. 2016, 6:27696). We performed RNA Microarrays in OCI-AML3 cells treated with C-82, ABT-199, or the combination and found that both C-82 and the combination downregulated multiple genes, including Rac1. It was recently reported that inhibition of Rac1 by the pharmacological Rac1 inhibitor ZINC69391 decreased MCL-1 expression in AML cell line HL-60 cells (Cabrera M et al, Oncotarget. 2017, 8:98509). We therefore hypothesized that inhibiting β-catenin by C-82 may potentiate BCL-2 inhibitor ABT-199 via downregulating Rac1/MCL-1. To investigate the effects of simultaneously targeting β-catenin and BCL-2, we treated AML cell lines and primary patient samples with C-82 and ABT-199 and found that inhibition of Wnt/β-catenin signaling significantly enhanced the potency of ABT-199 in AML cell lines, even when AML cells were co-cultured with mesenchymal stromal cells (MSCs). The combination of C-82 and ABT-199 also synergistically killed primary AML cells (P<0.001 vs control, C-82, and ABT-199) in 10 out of 11 samples (CI=0.394±0.063, n=10). This synergy was also shown when AML cells were co-cultured with MSCs (P<0.001 vs control, C-82, and ABT-199) in all 11 samples (CI=0.390±0.065, n=11). Importantly, the combination also synergistically killed CD34+ AML stem/progenitor cells cultured alone or co-cultured with MSCs. To examine the effect of C-82 and ABT-199 combination in vivo, we generated a patient-derived xenograft (PDX) model from an AML patient who had mutations in NPM1, FLT3 (FLT3-ITD), TET2, DNMT3A, and WT1 genes and a complex karyotype. The combination synergistically killed the PDX cells in vitro even under MSC co-culture conditions. After PDX cells had engrafted in NSG (NOD-SCID IL2Rgnull) mice, the mice were randomized into 4 groups (n=10/group) and treated with vehicle, C-82 (80 mg/kg, daily i.p injection), ABT-199 (100 mg/kg, daily oral gavage), or the combination for 30 days. Results showed that all treatments decreased circulating blasts (P=0.009 for C-82, P<0.0001 for ABT-199 and the combination) and that the combination was more effective than each single agent (P<0.001 vs C-82 or ABT-199) at 2 weeks of therapy. The combination also significantly decreased the leukemia burden in mouse spleens compared with controls (P=0.0046) and single agent treated groups (P=0.032 or P=0.020 vs C-82 or ABT-199, respectively) at the end of the treatment. However, the combination did not prolong survival time, likely in part due to toxicity. Dose modifications are ongoing. These results suggest that targeting Wnt/β-catenin and BCL-2, both essential for AML cell and stem cell survival, has synergistic activity via Rac1-mediated MCL-1 inhibition and could be developed into a novel combinatorial therapy for AML. Disclosures Andreeff: SentiBio: Equity Ownership; Oncolyze: Equity Ownership; Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Consultancy; Amgen: Consultancy, Research Funding; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Reata: Equity Ownership; Astra Zeneca: Research Funding; Celgene: Consultancy; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer . Carter:novartis: Research Funding; AstraZeneca: Research Funding.

scholarly journals Expression and in Vitro Binding of Protease Activated Receptor1 (PAR1) with Novel Anti-PAR1 Molecules: Data on Fresh Myeloma Marrow Plasma Cells and Human Myeloma Cell Lines

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4440-4440
Author(s):  
Meral Beksac ◽  
Pinar Ataca ◽  
Berna Atesagaoglu ◽  
Klara Dalva ◽  
Andry Nur Hidayat ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Lower Percentage ◽  
Advisory Committees ◽  
In Silico Docking ◽  
Human Myeloma Cell

Abstract Introduction and Aim: Myeloma plasma cells are dependent on stromal support which is mediated through cell adhesion. Heparanase activity has been shown to be associated with aggressive behavior or Bortezomib resistance and can lead to increased levels of proteases as well as shedding of heparan sulfate proteoglycan syndecan-1(CD138) from myeloma cells. We have recently published in vivo anti-myeloma effects of low molecular weight heparin (Beksac et al Acta Haematol 2015). Protease activated Receptor (PAR1) is a thrombin receptor. PAR1 gene and antigen expression on myeloma patient samples and cell lines (HMCL) has been recently reported by University of Arkansas (UAMS) group (Tian et al ASH 2011). They were able to find HMCLs H929, U266, JJN3 to express PAR1. Also expression was found to be highest among patients with 5q amplification where the PAR1 gene is located. Patients and Methods: We analyzed PAR1 expression (WEDE15 PE, Beckman Coulter) by flow cytometry, on CD38+CD138+/-CD27+/- cells obtained from fresh patient bone marrow samples obtained either at diagnosis (n: 84)(NDMM) or relapse (n: 54)(RRMM) and were compared with marrow samples taken from patients without MM (n: 43). Our group in Ankara University had previously synthesized and published novel benzamide and phenyl acetamide derivatives. We performed an in silico docking analysis on these molecules, and eleven (TD10,TD12,TD12A,TD12B,TD13,TD14,TD14B,XT2,XT2B,XT5,XT11) were found to bind to PAR1. These molecules were screened using 72 hour MTT assay on primary and refractory cell lines (U266BR ,U266, JJN3BR, JJN3, H929R, OPM2, OPM2R, KMS28PE). Results: PAR1 expression was highest on platelets followed by myeloma plasma cells (0-81.9%) and did not correlate with ISS. PAR1 expression (Threshold: >2.5 % or >5%) could be detected in NDMM (35 % or 14%) and RRMM (31% or 19%) of patients (Table1). PAR1+CD38+138+ cells were more frequent among patients with lower percentage of plasma cells in RRMM group (2,98 ± 4,5 vs 1,93 ± 3,96, P=0.028) but not NDMM. PAR1 was similarly highly expressed on HCML. Two of the novel PAR1 binding molecules (XT5 and XT2B) were found to have the lowest IC50. The IC50 were similar for all HMCLs, primary and refractory, with XT5. With XT2B the IC50 was less (U266) or higher (JJN3) or similar (OPM2) for refractory compared to the primary HMCL. PAR1 expression and anti-myeloma IC50 values of cell lines are summarized in Table 2. Conclusion: PAR1 expression is detectable at very low or very high percentages on CD138+plasma cells. Expression is higher on cells with CD27 expression (patient samples) or lacking CD27 (HMCL). Inverse correlation between PAR1 expression and plasma cell percentage among myeloma patients is detected among RRMM but not on NDMM samples. This finding may point to expression of PAR1 on quiescent plasma progenitors as suggested by Tian et al previously. The intensity or frequency of PAR1 expression on HMCL did not influence the anti-myeloma effects of these novel molecules. PAR1 binding molecules, in particular XT5, are promising as they are effective even on Bortezomib refractory HCML. However their mechanism of action and the role of PAR1 require further investigations. This study has been supported by a research grant from Turkish Academy of Sciences. Table 1. Frequency of PAR1 expression (> 2.5 %) on total plasma cells (CD38+138+) and on quiescent plasma cells (CD38+138+27+) Control (n=43) NDMM (n=84) RRMM (n=54) P CD38+138+ (%) 0,56± 0,66 4,48 ± 7,67 5,44 ± 12,13 0,007 PAR1+ among CD38+138 (%) 6,18 ± 13,14 4,14 ± 11,00 3,42 ± 8,81 0,394 PAR1+ among CD38+138+27+(%) 5,44 ± 12,13 3,42 ± 8,81 3,58 ± 8,57 0,207 Table 1. Comparison of Flow Cytometric PAR1 expression and IC50 (in uM after 72 hours)of the two novel molecules on three Human Myeloma Cell Lines. H929 RPMI8221 U266 IC50 XT2B 33.9 >100 34.3 IC50 XT5 8.12 5.45 9.77 CD38+138+ (total%) 85 % 75 % 80 % PAR1% and (MFI) within CD38+138+ 83 %(13,6) 90 % (2,1) 85 % (2,1) Disclosures Beksac: Celgene: Consultancy, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Off Label Use: Elotuzumab is an investigational agent being studied for the treatment of multiple myeloma.. Usmani:Millennium: Honoraria, Speakers Bureau; Sanofi: Honoraria, Research Funding; Onyx: Honoraria, Research Funding, Speakers Bureau; Celgene: Honoraria, Speakers Bureau; Array BioPharma: Honoraria, Research Funding; Pharmacyclics: Research Funding; Janssen Oncology: Honoraria, Research Funding. Tian:University of Arkansas for Medical Sciecnes: Employment.

scholarly journals In Vitro and inVivo Activity of Melflufen in Amyloidosis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3100-3100 ◽  
Author(s):  
Ken Flanagan ◽  
Muntasir M Majumder ◽  
Romika Kumari ◽  
Juho Miettinen ◽  
Ana Slipicevic ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Cell Lines ◽  
Plasma Cell ◽  
Light Chain ◽  
Research Funding ◽  
Plasma Cells ◽  
Al Amyloidosis ◽  
Advisory Committees

Background: Immunoglobulin light-chain (AL) amyloidosis is a rare disease caused by plasma cell secretion of misfolded light chains that assemble as amyloid fibrils and deposit on vital organs including the heart and kidneys, causing organ dysfunction. Plasma cell directed therapeutics, aimed at preferentially eliminating the clonal population of amyloidogenic cells in bone marrow are expected to reduce production of toxic light chain and alleviate deposition of amyloid thereby restoring healthy organ function. Melphalan flufenamide ethyl ester, melflufen, is a peptidase potentiated alkylating agent with potent toxicity in myeloma cells. Melflufen is highly lipophilic, permitting rapid cellular uptake, and is subsequently enzymatically cleaved by aminopeptidases within cells resulting in augmented intracellular concentrations of toxic molecules, providing a more targeted and localized treatment. Previous data demonstrating multiple myeloma plasma cell sensitivity for melflufen suggests that the drug might be useful to directly eliminate amyloidogenic plasma cells, thereby reducing the amyloid load in patients. Furthermore, the increased intracellular concentrations of melflufen in myeloma cells indicates a potential reduction in systemic toxicity in patients, an important factor in the fragile amyloidosis patient population. To assess potential efficacy in amyloidosis patients and to explore the mechanism of action, we examined effects of melflufen on amyloidogenic plasma cells invitro and invivo. Methods: Cellular toxicity and apoptosis were measured in response to either melflufen or melphalan in multiple malignant human plasma cell lines, including the amyloidosis patient derived light chain secreting ALMC-1 and ALMC-2 cells, as well as primary bone marrow cells from AL amyloidosis patients, using annexin V and live/dead cell staining by multicolor flow cytometry, and measurement of cleaved caspases. Lambda light chain was measured in supernatant by ELISA, and intracellular levels were detected by flow cytometry. To assess efficacy of melflufen in vivo, the light chain secreting human myeloma cell line, JJN3, was transduced with luciferase and adoptively transferred into NSG mice. Cell death in response to melflufen or melphalan was measured by in vivo bioluminescence, and serum light chain was monitored. Results: Melflufen demonstrated increased potency against multiple myeloma cell lines compared to melphalan, inducing malignant plasma cell death at lower doses on established light chain secreting plasma cell lines. While ALMC-1 cells were sensitive to both melphalan and melflufen, the IC50 for melphalan at 960 nM was approximately 3-fold higher than melflufen (334 nM). However, ALMC-2 cells were relatively insensitive to melphalan (12600 nM), but maintained a 100-fold increase in sensitivity to melflufen (121 nM). Furthermore, while 40% of primary CD138+ plasma cells from patients with diagnosed AL amyloidosis responded to melflufen treatment in vitro, only 20% responded to melphalan with consistently superior IC50 values for melflufen (Figure 1). Light chain secreting cell lines and AL amyloidosis patient samples were further analyzed by single cell sequencing. We further examined differential effects on apoptosis and the unfolded protein response in vitro in response to either melflufen or melphalan. This is of particular interest in amyloidosis, where malignant antibody producing plasma cells possess an increased requirement for mechanisms to cope with the amplified load of unfolded protein and associated ER stress. As AL amyloidosis is ultimately a disease mediated by secretion of toxic immunoglobulin, we assessed the effects of melflufen on the production of light chain invitro, measuring a decrease in production of light chain in response to melflufen treatment. Finally, we took advantage of a recently described adoptive transfer mouse model of amyloidosis to assess the efficacy of melflufen and melphalan in eliminating amyloidogenic clones and reducing the levels of toxic serum light chain in vivo. Conclusions: These findings provide evidence that melflufen mediated toxicity, previously described in myeloma cells, extends to amyloidogenic plasma cells and further affects the ability of these cells to produce and secrete toxic light chain. This data supports the rationale for the evaluation of melflufen in patients with AL amyloidosis. Figure 1 Disclosures Flanagan: Oncopeptides AB: Employment. Slipicevic:Oncopeptides AB: Employment. Holstein:Celgene: Consultancy; Takeda: Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy; Genentech: Membership on an entity's Board of Directors or advisory committees; Sorrento: Consultancy. Lehmann:Oncopeptides AB: Employment. Nupponen:Oncopeptides AB: Employment. Heckman:Celgene: Research Funding; Novartis: Research Funding; Oncopeptides: Research Funding; Orion Pharma: Research Funding.

A NOVEL Aurora A Kinase INHIBITOR MLN8237 Induces Cytotoxicity and CELL Cycle Arrest IN MULTIPLE MYELOMA.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3830-3830
Author(s):  
Gullu Gorgun ◽  
Elisabetta Calabrese ◽  
Teru Hideshima ◽  
Jeffrey Ecsedy ◽  
Giada Bianchi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Board Of Directors ◽  
Cell Lines ◽  
Mitotic Spindle ◽  
Research Funding ◽  
Thymidine Incorporation ◽  
Aurora A Kinase ◽  
Advisory Committees

Abstract Abstract 3830 Poster Board III-766 Multiple myeloma (MM) is an incurable bone marrow derived plasma cell malignancy. Despite significant improvements in treating patients suffering from this disease, MM remains uniformly fatal due to intrinsic or acquired drug resistance. Thus, additional modalities for treating MM are required. Targeting cell cycle progression proteins provides such a novel treatment strategy. Here we assess the in vivo and in vitro anti-MM activity of MLN8237, a small molecule Aurora A kinase (AURKA) inhibitor. AURKA is a mitotic kinase that localizes to centrosomes and the proximal mitotic spindle, where it functions in mitotic spindle formation and in regulating chromatid congression and segregation. In MM, increased AURKA gene expression has been correlated with centrosome amplification and a worse prognosis; thus, inhibition of AURKA in MM may prove to be therapeutically beneficial. Here we show that AURKA protein is highly expressed in eight MM cell lines and primary patient MM cells. The affect of AURKA inhibition was examined using cytotoxicity (MTT viability) and proliferation (3[H]thymidine incorporation) assays after treatment of these cell lines and primary cells with MLN8237 (0.0001 μM – 4 μM) for 24, 48 and 72h Although there was no significant inhibition of cell viability and proliferation at 24h, a marked effect on both viability and proliferation occurred after 48 and 72h treatment at concentrations as low as 0.01 μM. Moreover, MLN8237 inhibits cell growth and proliferation of primary MM cells and cell lines even in the presence of bone marrow stromal cells (BMSCs) or cytokines IL-6 and IGF1. Similar experiments revealed that MLN8237 did not induce cytotoxicity in normal peripheral blood mononuclear cells (PBMCs) as measured by MTT assay, but did inhibit proliferation at 48 and 72h, as measured by the 3[H]thymidine incorporation assay. To delineate the mechanisms of cytotoxicity and growth inhibitory activity of MLN8237, apoptotic markers and cell cycle profiles were examined in both MM cell lines and primary MM cells. Annexin V and propidium iodide staining of MM cell lines cultured in the presence or absence of MLN8237 (1 μM) for 24, 48 and 72h demonstrated apoptosis, which was further confirmed by increased cleavage of PARP, capase-9, and caspase-3 by immunoblotting. In addition, MLN8237 upregulated p53-phospho (Ser 15) and tumor suppressor genes p21 and p27. Cell cycle analysis demonstrated that MLN8237 treatment induces an accumulation of tetraploid cells by abrogating G2/M progression. We next determined whether combining MLN8237 with conventional (melphalan, doxorubucin, dexamethasone) and other novel (VELCADE®) therapeutic agents elicited synergistic/additive anti-MM activity by isobologram analysis using CalcuSyn software. Combining MLN8237 with melphalan, dexamethasone, or VELCADE® induces synergistic/additive anti-MM activity against MM cell lines in vitro (p≤0.05, CI<1). To confirm in vivo anti-MM effects of MLN8237, MM.1S cells were injected s.c. into g-irradiated CB-17 SCID mice (n=40, 10 mice EA group). When tumors were measurable (>100 mm3), mice were treated with daily oral doses of vehicle alone or 7.5mg/kg, 15mg/kg, 30mg/kg MLN8237 for 21 days. Overall survival (defined as time between initiation of treatment and sacrifice or death) was compared in vehicle versus- MLN8237- treated mice by Kaplan-Meier method. Tumor burden was significantly reduced (p=0.02) and overall survival was significantly increased (p=0.02, log-rank test) in animals treated with 30mg/kg MLN8237. In vivo anti-MM effects of MLN8237 were further validated by performing TUNEL apoptosis-cell death assay in tumor tissues excised from control or treated animals. Importantly, a significant dose-related increase in apoptotic cells was observed in tumors from animals that received MLN8237 versus controls. These results suggest that MLN8237 represents a promising novel targeted therapy in MM. Disclosures: Ecsedy: Millennium Pharmaceutical: Employment. Munshi:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium: Research Funding; Novartis: Research Funding; Celgene: Research Funding.

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