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 Modeling Ponatinib Resistance in BCR-ABL1+ Cell Lines: Implications for Ponatinib Resistance in TKI-Resistant and TKI-naïve Patients

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4515-4515 ◽  
Author(s):  
Liu Lu ◽  
Verity Saunders ◽  
Chung H Kok ◽  
Tamara Leclercq ◽  
Timothy P Hughes ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Fold Increase ◽  
Resistant Line ◽  
Advisory Committees ◽  
Resistant Lines ◽  
Tki Resistance ◽  
T315i Mutation

Abstract Introduction: Tyrosine kinase inhibitor (TKI) resistance remains a major impediment to successful treatment of patients with chronic myeloid leukemia (CML). The 3rd generation TKI ponatinib (Pon), has been demonstrated to successfully overcome BCR-ABL1 kinase domain (KD) mutation based resistance including BCR-ABL1T315I, which inhibits the binding of all other available TKIs. Pon has demonstrated efficacy in CML patients who acquired resistance to other TKIs. However, little is known about the mechanisms which result in resistance to Pon. Here, modes of Pon resistance have been investigated invitro in both TKI naïve and treated BCR-ABL1+ cell lines. Methodology and Results: Pon resistance was generated in 4 BCR-ABL1+ cell lines 1) K562, 2) K562 DOX (increased ABCB1) 3) K562 DOX 55D –resistant to 55 nM dasatinib (Das) and 4) K562 T315I – T315I mutation developed in the presence of Das. Both Das treated cell lines have 3-4 fold higher expression of BCR-ABL1 mRNA compared to control lines. The K562 T315I line contains 44% T315I mutation whereas no KD mutation was found in the other lines. Parental Pon naive controls were maintained in parallel. Resistant cell lines (Table 1) were established by exposing the above cell lines to increasing concentrations of Pon for 660-900 days. The K562 T315IR resistant line demonstrated a >17 fold increase in IC50Pon compared to naïve control. While no expression of ABCB1 or ABCG2 was observed, BCR-ABL1 transcript level was raised 7 fold with a concomitant 4 fold increase in pBcr-Abl by western blot. In addition, the level of T315I in the resistant line increased from 44% to 81%, suggesting that increasing T315I% together with Bcr-Abl upregulation were the major resistant mechanisms in this line. The K562 DOX 55DR line demonstrated transient increases in BCR-ABL1 that reduced to basal levels concomitant with the development of the compound mutations G250E (71%) and E255K (64%). The IC50Pon was increased 47 fold compared to control, and high Das and imatinib (Im) IC50s confirmed the resistance to other TKIs (IC50Das: 1350 nM and IC50Im: 260 μM). In contrast, the Das naïve K562R and K562 DOXR resistant lines did not have demonstrable KD mutations. While 7AAD and annexin V staining confirmed Pon resistance, the IC50Pon was not higher in K562R and only 2 fold higher in K562 DOXR compared to controls, suggesting resistance to Pon was likely mediated by a Bcr-Abl independent mechanism. This was supported by a 2 fold reduction in pBcr-Abl and pSTAT5 in the Pon resistant lines compared to controls. Therefore, the expression levels of alternative pathway molecules were examined to determine possible mechanisms. Axl, a receptor tyrosine kinase has been previously associated with TKI resistance. A 7 fold increase in AXL mRNA was found in K562R line, and the K562 DOXR line demonstrated increased Axl protein. Importantly, after 72 hours incubation with Pon and the Axl inhibitor, R428, the viability of the two resistant lines were significantly reduced (Fig 1), suggesting Axl is likely critical in mediating Pon resistance. Furthermore, increased Axl was associated with increased cell adhesion in both lines, and adherence is a hallmark of Axl expression. Conclusion: As Pon binds to native Bcr-Abl stronger than to Bcr-AblT315I, an increased proportion of T315I reduced sensitivity to Pon in vitro. Pon exposure in a cell line predisposed to KD mutations resulted in the development of the compound mutations G250E and E255K, which rendered the cells insensitive to all tested TKIs. Interestingly the mode of resistance in the two Das naïve lines appears Bcr-Abl independent, and is likely mediated by Axl. While limited to in vitro studies these findings suggest that in the setting of prior TKI exposure, mutations are likely to be a primary form of Pon resistance. In the TKI naïve setting Bcr-Abl independent modes of resistance may be more likely suggesting combination therapeutic approaches may be warranted to overcome or prevent Pon resistance in the high risk setting. Abstract 4515. Table 1: Summary of resistance mechanisms detected in 4 cell lines. Pon culture condition (nM) Pon resistance Bcr-Abl dependent/ independent BCR-ABL1 mRNA(fold ↑ ) pBcr-Abl & pSTAT5(fold ↑ ) KD mutation IC50Pon(fold ↑ ) AXL mRNA(fold ↑ ) ↑ cell surface Axl Adherence K562 T315IR 100 DP 7 4 T315I >17 5 ✖ ✖ K562 DOX 55DR 200 DP 1 1 G250E E255K 47 ✖ ✖ ✖ K562R 200 IDP 1 0.5 ✖ ✖ 7 ✖ ✓ K562 DOXR 200 IDP 3 0.5 ✖ 2 ✖ ✓ ✓ ✓= yes; x = no; ↑=increased; DP = dependent; IDP = independent. Disclosures 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; Pfizer: Membership on an entity's Board of Directors or advisory committees. White:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Ariad: Research Funding.

Response and Outcomes to Nilotinib at 24 Months in Imatinib-Resistant Chronic Myeloid Leukemia Patients in Chronic Phase (CML-CP) and Accelerated Phase (CML-AP) with and without BCR-ABL Mutations.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1130-1130 ◽  
Author(s):  
Jerald P. Radich ◽  
Giovanni Martinelli ◽  
Andreas Hochhaus ◽  
Enrico Gottardi ◽  
Simona Soverini ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Median Time ◽  
Research Funding ◽  
Philadelphia Chromosome ◽  
Chronic Phase ◽  
Cytogenetic Response ◽  
Molecular Response ◽  
Advisory Committees ◽  
Imatinib Resistant

Abstract Abstract 1130 Poster Board I-152 Background Nilotinib is a selective and potent BCR-ABL inhibitor, with in vitro activity against most BCR-ABL mutants (excluding T315I) indicated for the treatment of patients with Philadelphia chromosome positive (Ph+) CML in CPor AP resistant or -intolerant to prior therapy, including imatinib. In a previous analysis of nilotinib in patients with BCR-ABL mutations, mutations occurring at three specific amino acid residues (E255K/V, Y253H, and F359C/V) were shown to be associated with less favorable response to nilotinib. The current analysis is based on mature data with a minimum follow-up of 24-months for all patients. Outcomes of patients at 24 months were analyzed by mutation type. Methods Imatinib-resistant CML-CP (n = 200) and CML-AP (n = 93) patients were subdivided into the following mutational subsets: no mutation, sensitive mutations (including mutations with unknown in vitro IC50). or E255K/V, Y253H, or F359C/V mutations at baseline. Patients with mutations of unknown in vitro sensitivity were classified as sensitive in this analysis based on a previous finding that patients with these mutations responded similarly to nilotinib as patients with sensitive mutation. Patients with baseline T315I mutations were excluded from this analysis. Patient groups were analyzed for kinetics and durability of cytogenetic and molecular response to nilotinib, as well as event-free survival (EFS), defined as loss of hematologic or cytogenetic response, progression to AP/BC, discontinuation due to disease progression, or death, and overall survival (OS). Results In CML-CP and -AP patients with no mutation, sensitive mutations, or E255K/V, Y253H, or F359C/V mutations, hematologic, cytogenetic and molecular responses are provided in the Table. Overall, patients with no mutations responded similarly to patients with sensitive mutations, whereas patients with E255K/V, Y253H, or F359C/V mutations had less favorable responses. This correlation was observed in both CML-CP and CML-AP patients, respectively. Median time to CCyR was 3.3 months (range, 1.0–26.7) for CML-CP patients with no mutations, and 5.6 months (range, 0.9–22.1) for patients with sensitive mutations. At 24 months, CCyR was maintained in 74% of CML-CP patients with no mutation and in 84% of patients with sensitive mutations. One patient with CML-CP and an E255K mutation achieved CCyR at 25 months and maintained until last assessment at 30 months. Median time to MMR was similar at 5.6 months (range, 0.9–25.8) for CML-CP patients with no mutations and 5.6 months (range, 2.7–22.1) for patients with sensitive mutations. No patient with a less sensitive mutation achieved MMR. Median EFS and 24-month estimated OS rate are provided in the Table. Conclusions Imatinib-resistant CML-CP and CML-AP patients treated with nilotinib therapy with BCR-ABL mutations (excluding E255K/V, Y253H, or F359C/V) achieved rapid and durable cytogenetic responses, and estimated EFS and OS at 24 months similar to that of patients with no mutations, respectively. Patients with E255K/V, Y253H, or F359C/V mutations had lower and less-durable responses and shorter EFS than patients with sensitive mutations. Alternative therapies may be considered for patients with these uncommon mutations (E255K/V, Y253H, and F359C/V). Disclosures Radich: Novartis: Consultancy, Honoraria, Research Funding. Hochhaus:Novartis: Research Funding. Branford:Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding. Shou:Novartis: Employment. Haque:Novartis: Employment. Woodman:Novartis: Employment. Kantarjian:Novartis: Research Funding. Hughes:Bristol-Myers Squibb: Advisor, Honoraria, Research Funding; Novartis: Advisor, Honoraria, Research Funding. Kim: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; Wyeth: Research Funding. Saglio:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau.

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 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.

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.

scholarly journals The Allosteric Inhibitor ABL001 Is Susceptible to Resistance in Vitro Mediated By Overexpression of the Drug Efflux Transporters ABCB1 and ABCG2

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4841-4841 ◽  
Author(s):  
Laura N Eadie ◽  
Verity A Saunders ◽  
Tamara M Leclercq ◽  
Susan Branford ◽  
Deborah L White ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Kinase Domain ◽  
Efflux Transporters ◽  
Advisory Committees ◽  
Expression Levels ◽  
Respective Control ◽  
Allosteric Inhibitor

Abstract Tyrosine Kinase Inhibitors (TKIs) result in excellent responses in Chronic Myeloid Leukaemia (CML) patients. However, secondary resistance is observed in ~35% of patients, mostly due to Bcr-Abl kinase domain (KD) mutations. Overexpression of the efflux transporter ABCB1 is a known mediator of primary resistance to imatinib (IM) and nilotinib (NIL). ABL001, a potent allosteric inhibitor, binds to the myristate pocket of the Bcr-Abl KD. In this study, we modelled ABL001 resistance in vitro with particular focus on ABCB1 and ABCG2. ABL001 sensitivity was evaluated in BCR -ABL1 + cell lines K562, K562-Dox (ABCB1 overexpressing) and K562-ABCG2 (transfected with ABCG2 overexpression vector). Also, K562, K562-Dox and KU812 cells were made resistant to ABL001 by culturing long term in increasing concentrations of ABL001 (increased once >80% survival in culture for >10 days). Parental ABL001 naive cells were maintained in parallel culture. Initial onset of resistance was characterised by significantly increased IC50ABL001 based on AnnexinV/7-AAD cytotoxicity assays performed at each ABL001 dose escalation. Resistance mechanisms were interrogated during escalation and once 10 μM was reached (based on clinically achievable doses). Protein expression levels of efflux transporters ABCB1/ABCG2 were examined by flow cytometry; KD mutation sequencing and Bcr-Abl protein quantitation were performed. K562-Dox and K562-ABCG2 cells demonstrated significantly increased IC50ABL001 compared with parental K562 control cells: 256 and 299 nM respectively vs 23 nM, p<0.001 suggesting both ABCB1 and ABCG2 are important in ABL001 transport. Furthermore, resistance was reversible through use of specific inhibitors cyclosporin (ABCB1) and Ko143 (ABCG2). IC50ABL001 +inhibitors was comparable to that of control K562 cells: K562-Dox +cyclosporin=11 nM; K562-ABCG2 +Ko143=15 nM. A prior study identified ABCB1 overexpression as an initiator of resistance to IM and NIL (expression increased up to 7- and 5-fold in IM and NIL resistant cells compared with respective control cells p<0.002). In this study, expression levels of ABCB1 and ABCG2 were interrogated in 3 ABL001 resistant cell lines; results indicated overexpression of both transporters was integral in development of resistance. Up to 17- and 60-fold greater levels of ABCB1 and ABCG2 respectively was observed in resistant vs control cells (Table 1). No KD mutations were detected in early resistance intermediates; however, a myristate pocket mutation was detected in later stage KU812 ABL001 resistant cells (percentage mutation correlated with IC50ABL001). Table 1. ABL001 resistance characteristics in selected resistant intermediates IC50ABL001 (nM) Fold change (MFI) Bcr-Abl over expression KD mutation ABCB1 ABCG2 K562 Control 23 N N K562 500 nM ABL001 >2500 p <0.001 -1.4 p =0.003 48.3 p =0.007 Y N K562 10 μM ABL001 27, 800p <0.001 -2.3 p =0.535 60.3 p <0.001 Y N K562 10 μM ABL001 +Ko143 89 p <0.001 K562-Dox Control 256 N N K562-Dox 500 nM ABL001 2393 p <0.001 2.3 p <0.001 -2.9 p <0.001 N N K562-Dox 500 nM ABL001 +cyclosporin 17 p <0.001 K562-Dox 10 μM ABL001 >50, 000 p <0.001 -1.6 p <0.001 -0.1 p <0.001 N N KU812 Control 2.7 N N KU812 5 nM ABL001 6.4 p <0.001 2.2 p =0.008 4.0 p =0.002 Y N KU812 10 μM ABL001 33, 300 p <0.001 8.1 p =0.003 11.0 p =0.010 Y Y (90%) p-value: resistant vs respective control; n>3; N=No; Y=Yes While further in vitro and in vivo studies will determine the clinical relevance of efflux-mediated resistance to ABL001 mono- and combination therapy, our preclinical data provide evidence that kinase domain mutations may not be the predominant cause of ABL001 resistance; drug transporters likely play an important role as well. Susceptibility to ABCB1 overexpression is well recognised for IM and NIL resistance and we now show it is also relevant for ABL001. ABCG2-mediated resistance has not been observed with NIL or IM but is clearly important for ABL001. ABL001 treatment induced ABCG2 overexpression in two cell lines; both have negligible basal expression. ABCG2 overexpression preceded Bcr-Abl overexpression and mutation emergence. Importantly, ABL001 resistance was completely reversible in the presence of the ABCG2 inhibitor Ko143 (K562 10 μM ABL001 cells). Given the lack of strong evidence for ABCG2-mediated transport of NIL or IM at clinically relevant concentrations, our data provide a strong rationale for the use of ABL001 in combination with either of these TKIs. Disclosures Branford: Ariad: Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Qiagen: Membership on an entity's Board of Directors or advisory committees. White:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding. Hughes:ARIAD: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

scholarly journals Acid Ceramidase (ASAH1) Mediates Intrinsic and Intercellular Transfer of Proteasome Inhibitor Resistance in Multiple Myeloma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1206-1206
Author(s):  
Ryan T Bishop ◽  
Tao Li ◽  
Raghunandan R Alugubelli ◽  
Oliver Hampton ◽  
Ariosto Siqueira Silva ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Resistant Cell ◽  
Clinical Samples ◽  
In Vivo Model ◽  
Acid Ceramidase ◽  
Advisory Committees

Abstract INTRODUCTION: Despite proteasome inhibitors (PIs) improving multiple MM (MM) outcomes, patients often become resistant. Identifying mechanisms of resistance with translational potential are an urgent unmet clinical need. Preliminary studies from our group have identified that the therapeutically targetable acid ceramidase, ASAH1, is a key mediator of PI resistance and its presence in extracellular vesicles (EVs) derived from resistant MM cells, confers PI resistance on drug naïve MM cells. METHODS: Nanosight technology, transmission electron microscopy and immunoblot were used to define EVs. Viability and apoptosis assays were used to determine the effects of EVs and inhibitors on resistance acquisition/sensitization to PIs. LC-MS was used to interrogate EV cargo contents. Clinical relevance of ASAH1 was determined in multiple human data cohorts (M2GEN and MMRF CoMMpass). Genetic (shRNA) and pharmacological (ceranib-2) approaches were used to assess the role of ASAH1 mechanistically in vitro and in vivo using multiple isogenic naïve and PI resistant cell lines, patient derived CD138+ MM cells and NSG mouse models. RESULTS: Co-culture of sensitive MM cells with resistant MM-EVs alone significantly protected against PI cytotoxicity. Proteomic profiling revealed high levels of ASAH1 in EVs derived from PI resistant MM cells. Further, we observed ASAH1 is abundant in lysates of multiple PI resistant cell lines compared to their isogenic drug sensitive counterparts. In human datasets, high ASAH1 expression was noted in PI resistant MM patients compared to those newly diagnosed and correlated with significantly shorter survival times. Mechanistically, knockdown of ASAH1 led to reduced conversion of ceramide to sphingosine 1-phosphate (S1-P) and decreased expression/activity of the anti-apoptotic proteins MCL-1, BCL2 and BCL-xL and increases in pro-apoptotic BIM and NOXA. Notably, ASAH1 knockdown also significantly sensitized the cells to PI treatment and this effect was rescued by addition of exogenous S1-P. Pharmacological inhibition of ASAH1 with ceranib-2 also sensitized resistant cells to PI treatment and prevented EV mediated resistance transfer in vitro. This was recapitulated ex vivo with human clinical samples. Our orthotopic in vivo model using PI-resistant U266-PSR cells show that ceranib-2 is highly effective in limiting the growth of PI-resistant disease, protecting against MM induced bone disease, and increasing overall survival compared to both bortezomib and vehicle controls. CONCLUSION: We define the ceramidase ASAH1 as a novel, druggable target for the treatment of PI resistant MM. Disclosures Hampton: M2Gen: Current Employment. Siqueira Silva: AbbVie Inc.: Research Funding; Karyopharm Therapeutics Inc.: Research Funding. Shain: Janssen oncology: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi Genzyme: Consultancy, Speakers Bureau; Karyopharm Therapeutics Inc.: Honoraria, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy; GlaxoSmithLine, LLC: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Adaptive Biotechnologies Corporation: Consultancy, Speakers Bureau; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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