Proteomics as a Functional Tool in Evaluating Bortezomib Treatment and Drug Resistance Mechanism.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1805-1805
Author(s):  
Melissa G Ooi ◽  
Paul Dowling ◽  
Paul Richardson ◽  
Constantine S. Mitsiades ◽  
Martin Clynes ◽  
...  
Keyword(s):  
Cell Lines ◽  
Research Funding ◽  
Mass Spectra ◽  
Resistance Mechanism ◽  
Resistance Mechanisms ◽  
Resistant Cell ◽  
Sensitive Cell ◽  
Advisory Committees ◽  
Tandem Mass Spectra ◽  
Bortezomib Treatment

Abstract Abstract 1805 Poster Board I-831 Background Multiple Myeloma (MM) is an incurable plasma cell disorder. Bortezomib (PS-341, Velcade™) is a novel proteosome inhibitor with significant activity in multiple myeloma, although subsets of patients remain refractory to the activity of the drug. Hence, better characterization of the interactions of this drug with classical resistance mechanisms may identify improved treatment applications. Materials and methods: We examined resistance mechanism after bortezomib treatment by using paired sensitive and resistant cell lines (MM1.S and OPM-2) in MM. As the cell lines used were not isogenic, we wanted to ensure that the changes seen were related to drug treatment only by using a corresponding solid tumor counterpart; thyroid carcinoma cell lines (KAT-18 and WRO). By using paired cell lines from different tumor types and applying a stringent exclusion criteria, any proteins that are related to cell line or cancer type phenomenon would be excluded and any protein changes seen would be related to ‘real’ changes that were attributed to bortezomib treatment. Samples were analysed using 2D-DIGE, a technique based on pre-electrophoretic labelling of samples with one of three spectrally resolvable fluorescent CyDyes (Cy2, Cy3, and Cy5) allowing multiplexing of samples into the same gel. All 2D-DIGE images were scanned and collected on a Typhoon Fluorescent Imager. Pooled samples were used as an internal standard to quantify expression changes with statistical significance. Statistics and quantitation of protein expression were carried out initially using DeCyder Biological Variation Analysis (BVA) software before performing subsequent Extended Data Analysis (EDA). Proteins of interest were picked from preparative gels and full scan mass spectra were recorded in profile mode and tandem mass spectra in centroid mode. The peptides were identified using the information in the tandem mass spectra by searching against the SWISS PROT database using SEQUEST. Results 18 proteins have been identified to be differentially expressed in the sensitive cell line compared to the resistant cell lines. 6 proteins were up-regulated and 12 down-regulated in the sensitive cell lines compared to the resistant cell lines (t-test 0.02) and all proteins were >1.3-fold differentially expressed. Of the proteins that are significantly changed, caspase 3 is down-regulated in sensitive cell lines after bortezomib treatment and heat shock protein 70 (Hsp70) is up-regulated. Caspase 3 is a key apoptosis executioner and is known to be responsible for the proteolytic cleavage of many proteins when cells are treated with bortezomib. Hsp70 is a chaperone protein for both proapoptotic and antiapoptotic proteins; it has been demonstrated to be upregulated with bortezomib treatment. These two proteins correlate with what is known about how bortezomib affects cells and validates our results. Other proteins that were significantly changed are involved in transcriptional regulation. These proteins provide an interesting insight into how bortezomib treatment affects cellular processes and provide clues into resistance mechanisms. Conclusion As the field is now aware, bortezomib is more than a proteosome inhibitor and it affects other pathways in cellular function which would be difficult to evaluate using conventional analytical methods. Proteomics is a novel way to evaluate bortezomib's effect in cellular pathways and also drug resistance mechanisms. We are in the process of validating the other novel proteins identified in our screen using small molecule inhibitors to these proteins and/or knockdown studies to validate these proteins even further. Disclosures Richardson: Millenium: Membership on an entity's Board of Directors or advisory committees, Research Funding; MLNM: speakers bureau up to 7/1/09; Celgene: Membership on an entity's Board of Directors or advisory committees, speakers bureau up to 7/1/09. Mitsiades:Novartis Pharmaceuticals: Consultancy, Honoraria; Milllennium: Consultancy, Honoraria; Bristol-Myers Squibb : Consultancy, Honoraria; Merck &Co.: Consultancy, Honoraria; Kosan Pharmaceuticals : Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; PharmaMar: Patents & Royalties; Amgen: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis Pharmaceuticals: Research Funding. Anderson:Millenium: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

Novel Targeting Of Phospho-Marcks Overcomes Drug Resistance and Induces Anti-Tumor Activity In Preclinical Models Of Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 282-282 ◽  
Author(s):  
Yijun Yang ◽  
Manujendra N Saha ◽  
Yan Chen ◽  
Lugui Qiu ◽  
Donna E Reece ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Drug Resistance ◽  
Cell Lines ◽  
Research Funding ◽  
Resistant Cell ◽  
Drug Resistant ◽  
Sensitive Cell ◽  
Bortezomib Treatment ◽  
Cell Cycle Pathway ◽  
Rpmi 8226

Abstract Multiple myeloma (MM) remains incurable due to the development of a drug-resistant phenotype after prolonged therapy. Myristoylated alanine-rich C-kinase substrate (MARCKS) is a protein kinase C (PKC) substrate that plays an important role in cell adhesion, spreading and invasion. Our previous studies found that overexpression of phospho-MARCKS (pMARCKS) was detected in developed drug resistant MM cell lines (RPMI-8226 R5, MM.1R) relative to their parental drug sensitive cell lines (RPMI-8226S, MM.1S). We hypothesized that pMARCKS is involved in chemo- and novel drug resistance in MM. To further evaluate the drug resistance, we exposed both RPMI-8226 R5 and MM.1R cell lines to varying dosages of bortezomib, dexamethasone, doxorubicin, and lenalidomide. By MTT assay, both resistant cell lines were found to have significantly higher viability to all 4 drugs compared to their respective non-resistant lines. In addition, Western blot analysis showed increased pMARCKS expressions in all 3 bortezomib resistant cell lines 8226.BR, OPM2.BR, and ANBL-6.BR as compared to their respective bortezomib sensitive cell lines. We next acquired MM patient samples collected at diagnosis and at relapse after bortezomib treatment, and investigated their pMARCKS expression with immunoblotting analyses. The patient samples collected from relapse after bortezomib treatment had higher pMARCKS expression than those collected at diagnosis. Furthermore, we studied additional 3 primary MM patient samples with high pMARCKS expressions and 3 with low expressions for their vaibility after a 36 hour bortezomib treatment, and found that the samples with high pMARCKS expressions were more resistant to bortezomib than those with low pMARCKS expressions (mean IC50 of 7.1 nM and mean IC50 of 4.8 nM, respectively; p = 0.042). Importantly, combination of a low dosage of bortezomib (5.0 nM) with either 2.5 uM or 5.0 uM of enzastaurin (an inhibitor of phospho-PKC), displayed a synergistic cytotoxicity on myeloma cells with high pMARCKS expressions. To further elucidate the role of pMARCKS in drug resistance, we knocked down pMARCKS expression by transfecting siMARCKS into 8226 R5 and MM.1R cells. Following the knockdown, both cell lines had significantly lower viability after treatment with either bortezomib, dexamethasone, doxorubicin, or lenolidomide, in comparison to empty vector controls. FACS analysis and annexin V assay of the knockdown cells and the control cells from both cell lines showed significantly induction of G1/S cell cycle arrest and apoptosis in the knockdown cells. The immunoprecipitation (IP) and chromatin immunoprecipitation (ChIP) DNA-qPCR analysis further demonstrated that pMARCKS regulates SKP2 expression through binding with E2F1, mediating SKP2/p27Kip1 cell cycle pathway. Finally, we investigated the effect of inhibition of pMARCKS in a 8226 R5 xenograft model of SCID mice. Mice injected with shMARCKS-transfected 8226 R5 cells and received bortezomib showed significant retardation of tumor growth and prolonged survival compared to the control groups. Taken together, our data indicate that pMARCKS is constitutively activated in resistant and relapsed MM cells and contributes to drug resistance by regulating E2F1 mediated SKP2/p27Kip1 cell cycle pathway, thus providing a preclinical rationale for targeting pMARCKS as a promising approach in patients with refractory/relapsed MM. Disclosures: Reece: BMS: Research Funding; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Merck: Honoraria, Research Funding; Millennium: Research Funding; Novartis: Honoraria; Onyx: Honoraria.

The Interaction of Bortezomib with P-Gp, MRP-1 and BCRP Drug Transporters: Implications for Therapeutic Applications of Bortezomib in Advanced Multiple Myeloma and Other Neoplasias.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1729-1729
Author(s):  
Melissa G Ooi ◽  
Robert O'Connor ◽  
Jana Jakubikova ◽  
Justine Meiller ◽  
Steffen Klippel ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Multiple Myeloma ◽  
Cell Line ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Significant Activity ◽  
Cancer Resistance ◽  
Advisory Committees ◽  
Bortezomib Treatment

Abstract Abstract 1729 Poster Board I-755 Background Multidrug transporters are energy-dependent transmembrane proteins which can efflux a broad range of anticancer drugs and thereby play a role in resistance to the actions of substrate agents. Classically, three transporters, p-glycoprotein (Pgp; MDR-1; ABCB1), multidrug resistant protein-1 (MRP-1; ABCC1) and breast cancer resistance protein (BCRP; MXR; ABCG2), have been found to have the broadest substrate specificity and a strong correlation with drug resistance in vitro and in vivo in many models and forms of cancer. We have sought to characterize the interaction of bortezomib with these transporters and thereby explore the potential for these agents to play a role in resistance. Bortezomib is a novel proteosome inhibitor with significant activity in multiple myeloma, although subsets of patients remain refractory to the activity of the drug. Hence, better characterization of the interactions of this drug with classical resistance mechanisms may identify improved treatment applications. Methods and Results We investigated the role of these transporters by using isogenic cell line models which are resistant due to overexpression of a particular transporter: DLKP lung cancer cell line that overexpresses MRP-1; DLKP-A which overexpresses Pgp; and DLKP-SQ-Mitox which overexpresses BCRP. DLKP-A cells exhibited a 4.6-fold decrease in responsiveness to bortezomib compared to parental DLKP cells. In DLKP-SQ-Mitox, bortezomib-induced cytotoxicity was comparable to DLKP. When bortezomib was combined with elacridar, a Pgp and BCRP inhibitor, significant synergy was evident in DLKP-A (100% viable cells with single agent treatment versus 11% with the combination), but not DLKP-SQ-Mitox. Sulindac, an MRP-1 inhibitor, combined with bortezomib failed to produce any synergy in MRP-1 positive DLKP cells. Conversely, combination assays of Pgp substrate cytotoxics such as doxorubicin with Bortezomib were largely additive in nature. This indicates that bortezomib has little, if any, direct Pgp inhibitory activity, as combinations of a traditional Pgp inhibitor (such as elacridar) and doxorubicin would show marked synergy rather than just an additive effect in Pgp positive cells. To further characterize the extent of this interaction with Pgp, we conducted cytotoxicity assays in cell lines with varying levels of Pgp overexpression. NCI/Adr-res (ovarian cancer, high Pgp overexpression), RPMI-Dox40 (multiple myeloma, moderate Pgp overexpression) and A549-taxol (lung cancer, low Pgp overexpression). The combination of bortezomib and elacridar that produced the most synergy was in cell lines expressing moderate to high levels of Pgp expression. Cell lines with lower Pgp expression produced an additive cytotoxicity. We next examined whether bortezomib had any direct effect on Pgp expression. In RPMI-Dox40 cells, Pgp expression is reduced in a time-dependent manner with bortezomib treatment. Conclusions Our studies therefore show that bortezomib is a substrate for Pgp but not the other drug efflux pumps. In tumor cells expressing high levels of Pgp, the efficacy of bortezomib is synergistically enhanced by combinations with a Pgp inhibitor, while bortezomib treatment itself can reduce the expression of Pgp. This study suggests that in the subset of patients with advanced multiple myeloma or solid tumors which express high levels of Pgp, inhibition of its function could contribute to enhanced responsiveness to bortezomib. Disclosures Richardson: millenium: Membership on an entity's Board of Directors or advisory committees, Research Funding; celgene: Membership on an entity's Board of Directors or advisory committees, speakers bureau up to 7/1/09; MLNM: speakers bureau up to 7/1/09. Mitsiades:Millennium Pharmaceuticals : Consultancy, Honoraria; Novartis Pharmaceuticals : Consultancy, Honoraria; Bristol-Myers Squibb : Consultancy, Honoraria; Merck &Co: Consultancy, Honoraria; Kosan Pharmaceuticals : Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; PharmaMar: licensing royalties ; Amgen Pharmaceuticals: Research Funding; AVEO Pharma: Research Funding; EMD Serono : Research Funding; Sunesis Pharmaceuticals: Research Funding. Anderson:Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Biotest AG: Consultancy, Research Funding.

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 Identifying Mechanisms Associated with Venetoclax Resistance in Multiple Myeloma (MM)

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2668-2668
Author(s):  
Yuan Xiao Zhu ◽  
Laura Ann Bruins ◽  
Joseph Ahmann ◽  
Cecilia Bonolo De Campos ◽  
Esteban Braggio ◽  
...  
Keyword(s):  
Cell Line ◽  
Board Of Directors ◽  
Cell Lines ◽  
Acquired Resistance ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Sensitive Cell ◽  
Advisory Committees ◽  
Bcl2 Family ◽  
Family Regulation

Abstract Venetoclax (VTX) is a selective small-molecule inhibitor of BCL-2 that exhibits antitumoral activity against MM cells presenting lymphoid features and those with translocation t(11;14). Despite its impressive clinical activity, VTX therapy for a prolonged duration can lead to drug resistance. Therefore, it is important to understand the underlying mechanisms of resistance in order to develop strategies to prevent or overcome resistance. In the present study, we established four VTX resistant human myeloma cell lines (HMCLs) from four sensitive HMCLs, including three with t(11;14), in culture with a stepwise increase in treatment dose with VTX. To identify the molecular basis of acquired VTX resistance, whole exon sequencing (WES), mRNA-sequencing (mRNAseq), and protein expression assays were performed in the four isogenic VTX-sensitive/resistant HMCLs and three MM patients with samples collected before VTX administration and after clinical resistance to the drug. Compared with sensitive cell lines and patient samples collected before VTX administration, mRNAseq analysis identified downregulation of BIM and upregulation of BCLXL in both resistant cell lines and MM cells from relapse patients. Other transcriptional changes detected included upregulation of AURKA, BIRC3, BIRC5, and IL32. Enrichment analysis of differentially expressed genes suggested involvement of PI3K and MAPK signaling, likely associated with cytokines, growth factors (EGF, FGF and IGF family members), and receptor tyrosine kinase (EGF and FGF). Western blot analysis was performed to compare BCL2 family expression in resistant cell lines versus sensitive cell lines and it showed upregulation of BCL2 survival members (such as MCL-1 and BCLXL), and downregulation of pro-apoptotic BH3 members (such as BIM and PUMA). BIM expression was completely lost in one resistant cell line, and introduction of exogenous BIM into this cell line enhanced VTX sensitivity. Interestingly, BCL2 was upregulated in some resistant cell lines generated after a long-term treatment with VTX, suggesting BCL2 expression level may not be suitable as a marker of VTX sensitivity for acquired resistance. Unlike in CLL, BCL2 mutations were not identified through WES in any resistant cell lines or primary patient sample harvested after relapse. While 8 genes were mutated in two resistant samples , no clear mutational pattern emerged . Based on the above, we further tested some specific inhibitors in in vitro or ex vivo cell models to help understanding resistant mechanism and identify strategies to overcome VTX resistance. We found that inhibition of MCL-1, with the compound S68345, substantially enhanced VTX sensitivity in three resistant HMCLs and in primary cells from one relapsed MM patient. A BCLXL inhibitor (A155463) only significantly enhanced VTX sensitivity in one resistant cell line after co-treatment with VTX. Co-treatment of the other three resistant cell lines with VTX, S68345 and A155463 resulted in the most synergistic anti-myeloma activity, suggesting those cell lines are co-dependent on MCL-1, BCLXL, and BCL2 for survival, although they are more dependent on MCL-1. We also found that inhibition of PI3K signaling, IGF1, RTK (EGF and FGF) and AURKA significantly increased VTX sensitivity, partially through downregulation of MCL-1, and BCLXL, and upregulation of BIM. Conventional anti-MM drugs such as dexamethasone, bortezomib and lenalidomide, were shown to have little activity on augmenting VTX sensitivity in most resistant cell lines. In summary, we find that acquired resistance to VTX in MM is largely associated with BCL2 family regulation, including upregulation of survival members such as MCL-1, BCLXL, BCL2, and downregulation of pro-apoptotic members, especially BIM. Our study also indicates that upstream signaling involved in BCL2 family regulation during acquired resistance is likely related to cytokine, growth factor, and/or RTK-induced cell signaling such as PI3K. Co-inhibition of MCL-1, or BCLXL, as well as the upstream PI3K, RTK (FGF and EGF), IGF-1 mediated signaling were effective in overcoming VTX resistance. Disclosures Fonseca: Mayo Clinic in Arizona: Current Employment; Amgen: Consultancy; BMS: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Bayer: Consultancy; Janssen: Consultancy; Novartis: Consultancy; Pharmacyclics: Consultancy; Sanofi: Consultancy; Merck: Consultancy; Juno: Consultancy; Kite: Consultancy; Aduro: Consultancy; OncoTracker: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy; AbbVie: Consultancy; Patent: Prognosticaton of myeloma via FISH: Patents & Royalties; Scientific Advisory Board: Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Caris Life Sciences: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Tolinapant (ASTX660), a Non-Peptidomimetic Antagonist of cIAP1/2 and XIAP, and the HDAC Inhibitor Romidepsin Are Synergistic in in Vitro Models of T Cell Lymphoma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4356-4356
Author(s):  
John S Manavalan ◽  
Ipsita Pal ◽  
Aidan Pursley ◽  
George A. Ward ◽  
Tomoko Smyth ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Line ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
T Cell Lymphoma ◽  
Tnf Alpha ◽  
Sensitive Cell ◽  
Advisory Committees

Abstract Background: The PTCL are a heterogeneous group of non-Hodgkin lymphomas originating from mature T-lymphocytes. They are aggressive diseases, often resistant to conventional chemotherapy. Despite the fact that a number of new agents have been approved, treatment paradigms tailored to the biology of the disease have yet to emerge. Tolinapant (ASTX660) is a potent antagonist of both cellular and X-linked inhibitors of apoptosis proteins (cIAP1/2 and XIAP), and is presently in phase I/II trials in patients with advanced solid tumors and lymphomas (NCT02503423). IAP antagonists enhance tumor necrosis factor (TNF) receptor superfamily mediated apoptosis (Ward GA, et al. Mol Cancer Ther. 2018), are potent anti-tumor immune enhancers and induce markers of immunogenic cell death such as damage associated molecular patterns (DAMPs; Ye W, et al, Oncoimmunology, 2020). Objectives: We explored the sensitivity of a range of T-cell lymphoma (TCL) cell lines to tolinapant. We establish the synergy coefficient between tolinapant and the HDAC inhibitor, romidepsin, and interrogated the molecular basis of their synergistic interaction. Methods: A panel of human T-cell lymphoma cell lines were tested in proliferation assays (CellTiterGlo) for sensitivity to tolinapant in the presence or absence of 10ng/ml of TNF alpha. For combination studies, with tolinapant and romidepsin, each drug was tested at the IC10 and IC40 concentrations in the presence or absence of TNF alpha. Synergy scores using the Excess over Bliss (EOB) model were calculated using SynergyFinder (Aleksandr Ianevski et al; Nucleic Acids Research, 2020). Additionally, the effects of tolinapant and romidepsin on the IAPs and caspases were analyzed by western blots. TNFR1 receptor expression and induction of DAMPs were also analyzed by flow cytometry. Results: TCL Lines demonstrated varying sensitivities to tolinapant in the presence or absence of TNF alpha. The most sensitive cell lines, ALK+ ALCL and SUP-M2, had IC50 concentrations ranging from 200nM ± 100nM to 20nM ± 1nM in the absence or presence of TNF alpha, respectively, at 24, 48 and 72hrs, while a resistant CTCL cell line HH had an IC50 concentration of over 20mM, even in the presence of TNF alpha. Interestingly, using western blot analysis, we found that the presence of TNF alpha increased the levels of cIAP1 in the tolinapant sensitive SUP-M2 cell line, but not in the resistant HH cell line. However, there was a concentration dependent decrease in cIAP1 but not in XIAP in both cell lines treated with tolinapant. Flow cytometry analysis demonstrated that tolinapant increases the expression of TNFR1 and DAMPs in a dose dependent manner on the sensitive SUP-M2, but not in the resistant HH cells. In combination experiments, using the EOB model, tolinapant plus romidepsin was found to be synergistic in the absence of TNF alpha, at 36hrs, in both the sensitive cell line SUP-M2 and the resistant cell line HH. In the presence of TNF alpha, synergism was seen only in the sensitive cell line SUP-M2 and antagonistic in the HH cell line (Fig. 3). In the tolinapant plus romidepsin treated samples, cIAP1 levels decreased in the SUP-M2 cell line, in the absence of TNF alpha, however, addition of TNF alpha did not alter the levels of cIAP1 in the SUP-M2 cells. The cIAP1 levels decreased in the HH cells treated with the combination, in both the presence or absence of TNF alpha (Figure). Our findings indicate that the synergy of the tolinapant plus romidepsin is not dependent on the presence of TNF alpha. Conclusion: Tolinapant has demonstrated potent cytotoxic effects against a broad range of TCL lines both as a monotherapy and in combination with the HDAC Inhibitor, romidepsin. In in vitro studies, T cell lymphoma cell lines demonstrated varying sensitivity to tolinapant with certain cell lines being more resistant, even in the presence of TNF alpha. Interestingly, the addition of romidepsin appeared to overcome the intrinsic resistance to tolinapant in the absence of TNF alpha. These data provide the rationale to continue to explore the combination of tolinapant and romidepsin in vivo and to investigate additional combinations with T-cell specific agents (e.g. pralatrexate, belinostat, azacitidine and decitabine). Figure 1 Figure 1. Disclosures Smyth: Astex Pharmaceuticals: Current Employment. Sims: Astex Pharmaceuticals: Current Employment. Loughran: Kymera Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bioniz Therapeutics: Membership on an entity's Board of Directors or advisory committees; Keystone Nano: Membership on an entity's Board of Directors or advisory committees; Dren Bio: Membership on an entity's Board of Directors or advisory committees. Marchi: Kyowa Kirin: Honoraria; Myeloid Therapeutics: Honoraria; Astex: Research Funding; BMS: Research Funding; Merck: Research Funding; Kymera Therapeutics: Other: Scientific Advisor.

scholarly journals Inhibition of B-Cell Receptor Signaling Disrupts Cell Adhesion in Mantle Cell Lymphoma Via RAC2

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1369-1369
Author(s):  
Weige Wang ◽  
Franzen Carrie ◽  
Hui Guo ◽  
Jimmy Lee ◽  
Yan Li ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
B Cell ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Receptor ◽  
Resistant Cell ◽  
Advisory Committees ◽  
Tumor Tissues ◽  
Mantle Cell ◽  
Bcr Signaling

Abstract Background: B-cell receptor (BCR) signaling pathway is recognized as a crucial pathway for the pathogenesis of neoplastic B-cells. Inhibition of the BCR signaling and the downstream pathway is highly effective in B-cell malignancy through Bruton tyrosine kinase inhibition by ibrutinib. In addition to cell proliferation inhibition, ibrutinib disrupts cell adhesion between tumor and its microenvironment through unknown molecular mechanisms, resulting in peripheral lymphocytosis with accompanying lymphadenopathy reduction in patients who receive ibrutinib. Methods and materials: In an effort to elucidate the link between BCR signaling and cell adhesion phenotype, we first characterized ibrutinib sensitive and resistant mantle cell lymphoma (MCL) cell lines. We measured cell proliferation and cell growth, and correlated ibrutinib sensitivity with cell adhesion disruption. We then used RNA-sequencing to identify differential pathways between sensitive or resistant cell lines in response to ibrutinib treatment. We validated RNA-Seq findings using cell lines, as well as animal models and human primary MCL tumor tissues and cells. Results: We found that intrinsic sensitivities of MCL cell lines to ibrutinib correlated well with their cell adhesion phenotype. RNA-sequencing revealed that BCR and cell adhesion gene signatures were simultaneously down-regulated by ibrutinib in ibrutinib-sensitive but not ibrutinib-resistant cell lines. Among the differentially expressed genes in the BCR gene signature, we identified and validated that RAC2, a regulator of cell adhesion, was down-regulated at both RNA and protein levels by ibrutinib only in ibrutinib-sensitive cells. Physical association of RAC2 with BLNK, an early BCR pathway adaptor, was disrupted by ibrutinib uniquely in sensitive cells. RAC2 knockdown with siRNA impaired cell adhesion while RAC2 over-expression rescued ibrutinib-induced reduction in cell adhesion. In a xenograft mouse model, mice treated with ibrutinib demonstrated tumor growth retardation along with down-regulation in RAC2 protein expression. Using immunohistochemical staining, we demonstrated that RAC2 was expressed in ~65% primary MCL tumor tissues with majority of RAC2-positive tumors characterized as being the more aggressive subtypes. Finally, primary MCL cells treated with ibrutinib demonstrated reduced RAC2 that is accompanied by cell adhesion impairment. Conclusions: Our findings uncover a novel cross-talk between BCR signaling and cell adhesion. Ibrutinib inhibits cell adhesion via down-regulation of RAC2. Our study highlights the importance of RAC2 and cell adhesion in MCL pathogenesis and new drug development. Disclosures Wang: Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno: Research Funding; AstraZeneca: Consultancy, Research Funding; MoreHealth: Consultancy; Pharmacyclics: Honoraria, Research Funding; Novartis: Research Funding; Dava Oncology: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite Pharma: Research Funding; Acerta Pharma: 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.

scholarly journals Activity of the Phosphatidylinositol 3-Kinase (PI3K) Inhibitor, Copanlisib, As a Single Agent and in Combination with Carfilzomib in Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5665-5665
Author(s):  
Sarah M Larson ◽  
Mao Yu Peng ◽  
Andrae Vandross ◽  
Monica Mead ◽  
Zoe Fuchs ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Lines ◽  
Research Funding ◽  
Single Agent ◽  
Myeloma Cell ◽  
Resistant Cell ◽  
Cell Senescence ◽  
Sensitive Cell ◽  
Multiple Myeloma Cell

Abstract Background: The PI3K pathway signals for cell proliferation and survival in many malignancies including multiple myeloma. Copanlisib (BAY 80-6946) is a pan-class I PI3K inhibitor with preferential activity of the alpha and delta isoforms, of which the alpha isoform has particular importance in multiple myeloma. Here we demonstrate the pharmacological activity of copanlisib in multiple myeloma as a single agent and in combination with carfilzomib biomarker exploratory evaluation using phosphorylation of the S6 ribosomal protein (p-S6). Methods: 21 multiple myeloma cell lines were initially screened. Using an IC50 cut off of 100nM, 3 sensitive: NCI-H929, MM.1S, L-363 and 3 resistant: AMO-1, JJN3, COLO-677 were selected for further analysis. Apoptosis and cell senescence assays were done with each agent (copanlisib at 50nM and 100nM at 72 hours; carfilzomib at 2 nM and 20nM at 96 hours). Cell cycle analysis and induction of apoptosis were performed by FACS after propidium iodide or Annexin V FITC staining, respectively. Cellular senescencewas determined by measurement of β-galactosidase activity in cells treated for 96 hours. Combination studies utilized excess over highest single agent statistics (EOHSA) to evaluate potentiation. Reverse phase protein array (RPPA) was performed at baseline and post treatment for proteomics analysis with confirmatory western blot at 4 and 24 hours post treatment. Results: Copanlisib induced apoptosis and cell cycle arrest in the sensitive cell lines, but not the resistant cell lines. The cell senescence assays confirmed apoptosis rather than cell senescence as the mechanism of inhibition of proliferation. Pretreatment RPPA analysis demonstrated lower p-S6 levels in the sensitive cells lines compared to the resistant cell lines. Further, treatment with copanlisib resulted in a greater decrease in p-S6 in the sensitive cell lines than in the resistant cell lines, which was validated by western blot. Downstream pathway effects were confirmed by an increase in PDCD4 in the sensitive cell lines. Treatment with copanlisib and carfilzomib showed potentiation by EOHSA statistics and further decrease in p-S6 expression in the sensitive rather than resistant cell lines. Discussion: Copanlisib demonstrated single agent activity in human multiple myeloma cell lines, which is enhanced by the addition of carfilzomib. p-S6 levels may serve to select the most appropriate patient population to study combination of carfilzomib and copanlisib in relapsed/refractory multiple myeloma. With the choices of therapy available to patients with multiple myeloma there is a need for predictive biomarkers in order to better sequence therapies. Disclosures Larson: BMS: Consultancy. Slamon:Novartis: Consultancy, Honoraria, Research Funding; Biomarin: Consultancy, Honoraria; Pfizer: Honoraria, Research Funding; Eli Lilly: Consultancy; Syndax: Research Funding; Bayer: Consultancy.

scholarly journals Transcriptional Plasticity Compensates for Ikaros and Aiolos Proteasomal Degradation and Mediates Resistance to IMiDs in Multiple Myeloma (MM)

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 63-63
Author(s):  
Paola Neri ◽  
Ines Tagoug ◽  
Ranjan Maity ◽  
Caleb K Stein ◽  
Madison Kong ◽  
...  
Keyword(s):  
Family Member ◽  
Board Of Directors ◽  
Cell Lines ◽  
Transcriptional Repression ◽  
Research Funding ◽  
Proteasomal Degradation ◽  
Resistant Cell ◽  
Advisory Committees ◽  
Ets Family ◽  
Transcriptional Plasticity

Abstract Background: Immunoglobulin (IGH, IGL, IGK) and non-immunoglobulin (PVT1, TXNDC5, FAM46C, DUSP22, etc.) enhancers hijacking by variable genes (MYC, MAF, MAFB, CCND1/2/3, MMSET, IRF4) is a recognized oncogenic driver event in MM. However, the identity of the transcription factors (TFs) or transcriptional regulatory complexes binding and regulating the activity of these enhancers remains to be fully elucidated and may yield valuable therapeutic targets. As such the discovery of the BET family member BRD4 as the master histone acetyl mark reader at enhancers loci regulating MYC lead to promising therapeutic developments in MM and numerous other cancers. Immunomodulatory drugs (IMiDs) promote the proteasomal degradation of IKAROS (IKZF1) and AIOLOs (IKZF3) leading to the transcriptional repression of MYC and the suppression of MM cells survival and proliferation. However, acquired resistance to IMIDs and the loss of the transcriptional repression of MYC are nearly universal and occur in spite of sustained IKZF1/3 degradation suggesting that transcriptional rewiring may be sustaining hijacked enhancers activity and transcription of driver oncogenes. Methods and Results: In order to define how IMiDs repress MYC transcription, we first defined IKZF1, BRD4, the lysine acetyl transferase P300 and the mediator complex subunit MED1 mapping within the MM genome using ChIPseq. In MM cell lines (MM1S, RPMI8226, ARP1 and AMO1), IKZF1 predominantly mapped to intronic and intergenic loci which are typically enriched with enhancer and superenhancer elements. Indeed, IKZF1 mapping to the genome nearly completely (96.5%) overlapped that of P300, MED1 and BRD4 co-occupied enhancer and superenhancer loci. We also confirmed that in the MM1S sensitive cell lines IMiDs (lenalidomide 10 μM, 24h) exposure efficiently depleted IKZF1, BRD4, P300 and MED1 at enhancer loci with ensuing MYC (and MAF) downregulation. In contrast, in resistant cell lines (RPMI8226) and in spite efficient IKZF1 displacement, BRD4, P300 and MED1 were retained at the oncogenic enhancer (IGLL5) driving MYC (and MAF). These findings lead us to postulate that in IMiDs resistant cells retention of BRD4 and MED1 at oncogenic enhancers in the absence of IKZF1 likely results from rewiring of the TFs regulating MYC. To identify TFs that may co-localize with BRD4 and IKZF1, we analyzed the enrichment of DNA motifs at IKZF1and BRD4 co-occupied enhancers using the MEME suite motif-finding algorithms. This computational analysis revealed a strong enrichment at these MM enhancers of the GGAA motif recognized by the ETS family of TFs (P= 3.2 e-743) and other motifs boxes for the RUNX (P= 9.6 e-725), MYC/MYB ( P= 8.8 e-52) and interferon regulatory (IRF) (P= 3.1 e-293) TFs. We next confirmed that the ETS family TF ETV4 was indeed expressed in IMiDs resistant, but not sensitive, MM cell lines. ChiPseq occupancy profiles in IMiDs resistant RPMI8226 cell line revealed co-localization of ETV4 with IKZF1, P300 and BRD4. As predicted, lenalidomide treatment induced global depletion of IKZF1 but not ETV4 at BRD4 occupied enhancers in resistant cell lines (RPMI8226 and ARP1). Importantly, Cas9-mediated knock out of ETV4 in RPMI8226 cells sensitized them to lenalidomide with ensuing MYC downregulation and cell death. Confirming its role in MM, ETV4 transcript was indeed detectable in primary patients' samples in the CoMMpass data repository (ETV4 FPKM >1.0 in 112/724) and its expression was associated with significantly reduced survival outcomes (HR 0.64; P=0.0008). Similarly, high expression (top quartiles) of RUNX2 or MYB, TFs with enriched motifs at IKZF1 co-occupied enhancer loci, was also associated with decreased survival. Of note RNAseq analysis of paired patient samples pre- and post-IMiDs treatment (n=14 pairs) revealed significant upregulation of ETV4 at the time of acquired IMiDs resistance (7/14). Lastly transcriptome analysis of 101 patients enrolled in the RD arm (lenalidomide and dexamethasone) of the POLLUX trial (NCT02076009) confirmed the reduced survival of patients with top quartiles expression of ETV4 as well as MYB and RUNX2 (Fig.1) Conclusion: Transcriptional plasticity with expression of extra-lineage TFs such as the ETS family member ETV4 sustains oncogenic enhancers in MM overcoming IKAROS and AIOLOS dependency and promoting IMiDs resistance. Figure 1 Figure 1. Disclosures Neri: Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding. Soong: Jannsen: Employment. Chiu: Janssen: Employment. Bahlis: Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Pre-Clinical Evaluation of the PI3K-p110β/δ Inhibitor KA2237 in Mantle Cell Lymphoma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1837-1837 ◽  
Author(s):  
Shengjian Huang ◽  
Loretta J. Nastoupil ◽  
Hui Guo ◽  
Taylor Bell ◽  
Makhdum Ahmed ◽  
...  
Keyword(s):  
Cell Viability ◽  
Tumor Cells ◽  
Cell Lines ◽  
Research Funding ◽  
Synergistic Effects ◽  
Resistant Cell ◽  
Dependent Manner ◽  
Advisory Committees ◽  
Mantle Cell ◽  
Dose Dependent

Abstract Background: Mantle cell lymphoma (MCL) accounts for 6% of all non-Hodgkin lymphoma and is a therapeutic challenge. Phosphoinositide-3 kinase (PI3K) has been shown to be an alternative survival pathway in relapsed/refractory MCL. KA2237 (designed by Karus Therapeutics Ltd, Oxfordshire, United Kingdom) is a dual inhibitor of the class I beta and delta isoforms of the 110 kDa catalytic subunit of PI3K. By selectively targeting PI3K-beta and -delta isoforms and preventing their activation, KA2237 may decrease proliferation and induce cell death in susceptible tumor cells. Methods: We assessed the effects of KA2237 on the in vitro cell proliferation of both ibrutinib-sensitive (Mino, Jeko-1, and Rec-1) and primary ibrutinib-resistant (Z-138 and Maver-1) cell lines, and acquired ibrutinib-resistant MCL cell line, Jeko-R. We also tested the viability of patient-derived xenograft (PDX) tumor cells to KA2237. We compared the efficacy of KA2237 with two other commercial PI3K inhibitors, duvelisib (IPI-145, Selleck) and idelalisib (Cal-101, Selleck). Also, we paired these three inhibitors (KA2237, duvelisib and idelalisib) each with ibrutinib to evaluate the potential synergistic effects of these combinations. Lastly, we also tested in vivo efficacy of KA2237 and its combination with ibrutinib in PDX tumor cells. Results: KA2237 inhibited cell proliferation in both ibrutinib-sensitive and ibrutinib-resistant cell lines in a dose-dependent and time-dependent manner. For Mino and Jeko-1, the IC50 was 4.8 uM and 2.9 uM and for Z-138 and Maver-1 cell lines, the IC50 was 0.6 uM and 0.1 uM, respectively. KA2237 also decreased cell viability of ibrutinib-sensitive and ibrutinib-resistant MCL PDX tumor cells. However, KA2237 did not decrease the cell viability of normal human peripheral blood mono-nuclear cells. KA2237 arrested phase G0/G1 in Rec-1 and Jeko-R cell lines. We detected the expression of PI3K isoforms in MCL, finding higher expression of PI3K β and δ in MCL-resistant cell lines as compared with sensitive cell lines. We found that KA2237 induced MCL cell apoptosis in a time-dependent and dose-dependent manner. In comparison with duvelisib and idelalisib, KA2237 achieved greater inhibition of cell viability, cell apoptosis and cell cycle arrest. Furthermore, we found synergistic effects of KA2237 and ibrutinib combination in several MCL cell lines and in PDX models. In an ibrutinib-resistant PDX model, KA2237 treated mice reduced tumor burden significantly compared with vehicle control, and higher tumor growth inhibition was achieved as compared with ibrutinib. Conclusion: The novel PI3K inhibitor, KA2237 may be a potential candidate for MCL therapy, especially in the ibrutinib-resistant cases. Disclosures Wang: Acerta Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno Therapeutics: Research Funding; Pharmacyclics: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx: Research Funding; BeiGene: Research Funding; Asana BioSciences: Research Funding; Kite Pharma: Research Funding; Celgene: Research Funding.

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