At-101 Induces Apoptosis Waldenström Macroglobulinemia Cells Resistant to Bortezomib.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2861-2861
Author(s):  
Kasyapa Chitta ◽  
Kiersten M. Miles ◽  
Pushpankur Ghoshal ◽  
Leighton Stein ◽  
Morton Coleman ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Line ◽  
Board Of Directors ◽  
Therapeutic Potential ◽  
Cross Resistance ◽  
Advisory Committees ◽  
Cellular Activation ◽  
Potent Inducer ◽  
Resistant Cells

Abstract Abstract 2861 Poster Board II-837 Bcl-2 protein family has the unique capability to balance between the cell survival and death by regulating the expression of its individual members. AT-101 is a BH3 mimetic and a potent inducer of noxa and puma, the natural ligands of BH3 family proteins. It is known to bind and inhibit the anti-apoptotic functions of Bcl-2 family members Bcl-2 and Bcl-XL and Mcl-1. In vitro it has been shown to induce apoptosis in several tumor models systems including multiple myeloma. In this report we investigated the effect of AT-101 on a model cell line, BCWM.1 (a known WM cell line, BCWM.1/WT), representing Waldenström Macroglobulinemia. This disease is characterized by the presence of lymphoplasmacytic cells in the bone marrow and the secretion of IgM monoclonal protein into the serum. Several conventional therapies are available but the disease remains incurable. Therefore there remains a need to develop new therapies for this orphan disease. Recently, bortezomib (a proteasomal inhibitor) has shown promising anti-WM activity with enhanced responses when combined with traditional therapies. But continued treatment with bortezomib result in the development of resistance in the clinic. We developed an in vitro model of bortezomib resistance from BCWM.1 (hereafter referred as BCWM.1/BR). These cells also developed cross resistance to conventional therapies used for WM such as fludarabine and doxorubicin. Biological characterization of this cell line demonstrated Bcl-2 as a potentially important therapeutic target. We therefore assessed the effect of AT-101 to identify preclinically if this could be a potential clinical strategy in future. AT-101 induced a dose and time dependent inhibition in the viability of both BCWM.1/WT as well as BCWM.1/BR cells. Cell death was observed at as low as 1uM concentration of AT-101 and at 10uM a maximum of 50-70% death was observed by 24h. While BCWM.1/WT cells showed a significant death at 12h, treatment with AT-101 induced cell death in BCWM.1/BR cells as early as 6h. These results indicate that AT-101 induced a potent and quick inhibition in viability in BCWM.1/BR cells as compared to their parental wild type cells. Investigation into the mechanism of cell death showed that AT-101 induced apoptosis in a mitochondrial dependent pathway in these cells. A comparative analysis of the signal transduction pathways operated in BCWM.1/WT and BCWM.1/BR cells showed that many of the cellular activation and survival pathways such as AKT, ERK1/2 that are present in BCWM.1 cells are inhibited in the resistant cells. Interestingly, BCWM.1/BR cells expressed a fivefold increase in the Bcl-2 protein as compared to BCWM.1/WT cells suggesting a Bcl-2 dependent survival of these cells in the absence of other cellular activation and survival signals. Increased susceptibility of BCWM.1/BR cells to AT-101 thus can be understood to be a direct consequence of an increased expression of Bcl-2 and a dependence of the resistant cells on Bcl-2 family of anti-apoptotic proteins for their survival. Results presented in this report suggest that AT-101 has a unique therapeutic potential against Waldenström Macroglobulinemia that is independent of resistance to bortezomib. These observations highlight bcl-2 as a potential target, and AT-101 as possible therapeutic avenue for WM patients. Disclosures: Chanan-Khan: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Immunogen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

scholarly journals Establishing a Novel Pipeline That Combines in-Silico Prediction with in-Vitro and Ex-Vivo Validation to Discover Secondary Drug Combinations Against Relapsed and/or Refractory Multiple Myeloma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1615-1615
Author(s):  
Sayak Chakravarti ◽  
Suman Mazumder ◽  
Harish Kumar ◽  
Neeraj Sharma ◽  
Ujjal Kumar Mukherjee ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Line ◽  
Board Of Directors ◽  
In Silico ◽  
Research Funding ◽  
Ex Vivo ◽  
Standard Of Care ◽  
In Silico Prediction ◽  
Advisory Committees

Abstract Multiple myeloma (MM) is the second-most common hematological malignancy in the US. MM is an incurable, age-dependent plasma cell neoplasm with a 5-year survival rate of less than 50%. Extensive inter-individual variation in response to standard-of-care drugs like proteasome inhibitors (PIs) and immunomodulatory drugs (IMiDs), drug resistance, and dose-limiting toxicities are critical problems for the treatment of MM. Clinical success in anti-myeloma treatment, therefore, warrants continuous development of novel combination therapy strategies with the explicit goal to improve the therapeutic efficacy by concomitantly targeting multiple signaling pathways. Previously, we have reported the development of an in-house computational pipeline called secDrug that applies greedy algorithm-based set-covering computational optimization method followed by a regularization technique to predict secondary drugs that can be repurposed as novel synergistic partners of standard-of-care drugs for the management of refractory/ resistant MM. Top among these secondary drugs (secDrugs) were the HSP90 inhibitor 17-AAG. In this study, we used 17-AAG as a proof of principle to establish a pipeline that integrates our in silico predictions with in vitro and ex vivo validation as well as multi-omics technologies to identify, validate, and characterize therapeutic agents that could be used either alone or in combination with standard-of-care drugs for the treatment of R/R MM patients (Figure 1). To screen and validate our in silico prediction results, we performed in vitro cytotoxicity assays using 17-AAG on a panel of human myeloma cell lines (HMCLs; in vitro model systems) that captures a wide range of biological and genetic heterogeneity representing the complexities encountered in clinical settings. These cell lines include HMCLs representing innate sensitive/resistance, >10 pairs of parental and clonally-derived PI- and IMiD-resistant pairs (P vs VR or LenR; representing acquired/emerging resistance/relapse), NRAS mutants which leads to the constitutive activation of oncogenic Ras signaling, and CRISPR-edited HSP90 knockdown cell line. Our results showed that 17-AAG has high synergistic activity in combination with PI in inducing apoptosis even in innate and acquired PI-resistant HMCLs and significantly reduces the effective dose of PI required to achieve IC 50 (Chou-Talalay's Dose Reduction Index or DRI 7±1.4). Moreover, 17-AAG+IMiD showed synergistic cell killing activity in clonally-derived IMiD resistant HMCL. Further, 17-AAG induced cell death was comparable with Hsp90 knockdown as evident from the cytotoxicity assay using PI and 17-AAG in combination in RPMI8226-wild type and RPMI-HSP90AA1 knocked down cell line. Notably, 17-AAG was strikingly effective against the NRAS-mutant cell line indicating an additional niche (NRas mutant myeloma) where 17-AAG could be most effective. Next, we performed RNA sequencing to elucidate the molecular mechanisms behind 17-AAG drug action, drug synergy, 17-AAG-induced cell death. Our gene expression profiling (GEP) followed by Ingenuity Pathway Analysis (IPA) analysis revealed protein ubiquitination, aryl hydrocarbon receptor signalling pathway as the top canonical pathways. 17-AAG induced apoptosis via mitochondrial mediated pathway in myeloma. 17-AAG exerts its cytotoxic effect by activating intrinsic pathway of apoptosis which we further confirmed through the increase in reactive oxygen species generation and decrease in mitochondrial membrane potential. 17-AAG was also effective in reducing the expression of hallmarks of MM such as p65/NF-kB, IRF4, c-Myc. Finally, we performed mass cytometry (CyTOF; Cytometry by time of flight) on primary bone-marrow cells (PMCs) from myeloma patients for further validation of proteomic signatures at the single-cell level. CyTOF analysis confirmed 17-AAG-induced cell death and key changes in MM-specific proteomic markers. 17-AAG treated PMCs showed elevated cleaved caspase levels and down-regulation of IRF4 and phospho-STAT3. GEP and CyTOF results were confirmed using immunoblotting assays. Together, our study demonstrates a unique pipeline for drug repositioning that has the potential to revolutionize clinical decision-making by minimizing the number of drugs required for discovering successful combination chemotherapy regimens against drug-resistant myeloma. Figure 1 Figure 1. Disclosures Kumar: BMS: Consultancy, Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Tenebio: Research Funding; Beigene: Consultancy; Oncopeptides: Consultancy; Antengene: Consultancy, Honoraria; Carsgen: Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Roche-Genentech: Consultancy, Research Funding; Bluebird Bio: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding.

Mutations in CRBN and Other Cereblon Pathway Genes Are Only Associated with Acquired Resistance to Immunomodulatory Drugs in a Subset of Patients and Cell Line Models

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 6-7
Author(s):  
Amy Barber ◽  
John R Jones ◽  
Harvey Che ◽  
Yann-Vai Le Bihan ◽  
Niels Weinhold ◽  
...  
Keyword(s):  
Cell Line ◽  
Board Of Directors ◽  
Research Funding ◽  
Cross Resistance ◽  
Immunomodulatory Drugs ◽  
Private Company ◽  
Advisory Committees ◽  
Data Set ◽  
Synonymous Mutations

Background: Immunomodulatory drugs (IMiDs) are the current backbone of standard and experimental combination myeloma therapies at all stages of disease, but the majority of patients eventually relapse. The mechanisms driving IMiD resistance are poorly understood. Previous studies looking for genetic drivers of resistance have looked at core members of the CRL4CRBN E3-ubiquitin ligase complex (CUL4-RBX1-DDB1-CRBN) and identified infrequent mutations and deletions in cereblon (CRBN), but at a rate that cannot account for resistance in the majority of patients. More recently several in vitro studies have identified novel regulators of cereblon activity including the COP9 signalosome, E2 ubiquitin conjugating enzymes, neddylation modifiers and additional IMiD neosubstrates. In this study paired presentation/relapse samples from newly diagnosed patients recruited to a clinical trial (UK NCRI Myeloma XI trial: NCT01554852) of largely IMiD-based therapies were used to investigate the role of mutations and deletions in all genes implicated in IMiD activity. For comparison, cell line models of resistance were generated in vitro. Methods: 56 patients who received IMiD induction therapy followed by either lenalidomide maintenance (n=30) or observation (n=26), and subsequently relapsed, underwent whole exome sequencing (WES) of CD138+ cells, median depth 122x for tumour samples and 58x for paired germline controls. Non-synonymous mutations and deletions present in tumour but not germline controls were considered. Cell line models were generated using the IMiD sensitive MM1s cell line. Cells were cultured in 10xGI50 concentrations of lenalidomide/pomalidomide alongside a control exposed to the same %DMSO. WES was carried out and non-synonymous mutations identified. Mutations present in the lenalidomide resistant (Len-R) and pomalidomide resistant (Pom-R) but not their relevant DMSO exposed control were considered. From recent publications a list of 42 genes (Figure 1) involved in cereblon pathway regulation and IMiD response was curated, termed "CRBN/IMiD genes". Mutations in CRBN/IMiD genes in the patient dataset and cell line models were examined. Results: In the patient data set 12/42 (28.6%) of the CRBN/IMiD genes were found to be mutated, with a total of 17 mutations in 14/56 (25%) patients identified. 9/17 (53%) were identified in patients who had received lenalidomide maintenance and 8/17 (47%) in the observation group. Importantly, in the patients receiving lenalidomide maintenance, 6 of the 9 (66.7%) mutations had a higher cancer clonal fraction (CCF) at relapse, suggesting they may have been selected for by exposure to treatment. Comparatively, in mutations identified in patients undergoing observation, only 3 of the 8 (37.5%) mutations had a higher CCF at relapse compared with presentation. The only deletion in CRBN/IMiD genes was in SETX, in one patient at relapse. Only one mutation or deletion was identified in CRBN itself, a missense mutation at relapse at g.3:3195148A>C, encoding a Cys326Gly sequence modification at the protein level. Interestingly, Cys326 is one of 4 cysteines in CRBN coordinating a single zinc ion to form a Zn finger motif, which stabilises the Thalidomide Binding Domain (TBD) of the protein, suggesting this mutation may have had functional significance. In the cell line models full resistance up to 100xGI50 concentrations was established by 12 weeks. The resistant cell lines had cross-resistance to the other IMiDs and comparable morphology, growth rates and responses to non-IMiD drugs as their sensitive counterpart. Resistant cells had reduced levels of CRBN mRNA and protein expression. Functional assays demonstrated that well characterised downstream effects of IMiD treatment were abrogated: transcription factors Ikaros and Aiolos not degraded and no downregulation of IRF4 mRNA. The Pom-R cell line had a mutation affecting a CRBN splice site 5' of exon 8. No other mutations or deletions in the 42 IMiD pathway genes were identified in either the Len-R or Pom-R lines. Conclusions: CRBN and other genes in the IMiD response pathway were mutated or deleted in around 25% of patients suggesting other mechanisms, for example epigenetic alterations, underlie resistance acquisition in a significant proportion. Models for both CRBN/IMiD gene mutated and unmutated resistant states have been generated and will be used to study mechanisms of IMiD resistance. Figure Disclosures Jones: Celgene: Honoraria, Research Funding. Che:Monte Rosa Therapeutics: Research Funding. Le Bihan:Monte Rosa Therapeutics: Research Funding. Wang:Monte Rosa Therapeutics: Research Funding. Kaiser:Bristol-Myers Squibb, Chugai, Janssen, Amgen, Takeda, Celgene, AbbVie, Karyopharm, GlaxoSmithKline: Consultancy; Janssen, Amgen, Celgene, Bristol-Myers Squibb, Takeda: Honoraria; Bristol-Myers Squibb/Celgene, Janssen, Karyopharm: Research Funding; Bristol-Myers Squibb, Takeda: Other: Travel expenses. Jackson:Takeda: Honoraria, Research Funding, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Gsk: Honoraria, Speakers Bureau; Celgene: Honoraria, Research Funding, Speakers Bureau. Davies:Celgene/BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotech: Honoraria; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees. Chopra:Apple Tree Life Sciences: Current Employment; Monte Rosa Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Research Funding. Morgan:GSK: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Janssen: Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Pawlyn:Takeda: Consultancy, Other: Travel expenses; Celgene: Consultancy, Honoraria, Other: Travel expenses; Janssen: Honoraria, Other: Travel expenses; Amgen: Consultancy, Other: Travel expenses.

Induced Resistance to Bortezomib in Preclinical Model of Waldenstrom Macroglobulinemia Is Associated with Bcl-2 Upregulation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4919-4919 ◽  
Author(s):  
Kasyapa Chitta ◽  
Jean-Gabriel Coignet ◽  
Sakina Sojar ◽  
Pushpankur Ghoshal ◽  
Kiersten M. Miles ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Cell Line ◽  
Board Of Directors ◽  
Induced Resistance ◽  
Wild Type Cell ◽  
Preclinical Model ◽  
Wild Type ◽  
Advisory Committees ◽  
Proteasomal Activity ◽  
Resistant Cells

Abstract Abstract 4919 Waldenstrom's macroglobulinemia (WM) is characterized by the presence of lymphoplasmacytic cells in the bone marrow and the secretion of IgM monoclonal antibody in the serum. Several conventional therapies are available but the disease remains incurable. Recently, bortezomib (a proteasomal inhibitor) has shown promising anti-WM activity with enhanced responses when combined with traditional therapies. Resistance to bortezomib therapy is an important event that is associated with continued treatment. In order to understand the mechanism of bortezomib resistance in WM we exposed BCWM.1 (a known WM cell line) in vitro to increasing concentrations of bortezomib over prolonged periods of time and isolated the bortezomib resistant clone (BCWM.1/BR). This clone was compared with its parent wild type cell line (BCWM.1/WT). Investigation to understand the susceptibility of BCWM.1/Br cells to various therapeutic agents showed that these cells are resistant to many of the agents such as melaphalan, fludarabine or doxorubicin. Interestingly, verapamil, a broad spectrum inhibitor of multidrug resistance, failed to reverse the resistance induced by bortezomib indicating that bortezomib resistance is not because of an activation of multidrug resistance in these cells. While BCWM.1/WT cells showed an IC50 of 7.8nM when treated for 72h with bortezomib, the BCWM.1/BR cells were not inhibited at any concentration of the compound up to 100nM. Furthermore, the cells with the acquired resistance showed a 4 fold increase in the proteasomal activity as measured by the release of a fluorescent product (7-Amino-4-methylcoumarin (AMC)) from its peptide substrate, suc-LLVY-AMC. Biochemical analysis further revealed that many of the proteasomal components are altered in BCWM.1/BR cells as compared to their parental control cells. Interestingly, protein levels of two of the proteasomal catalytic subunits, PSMB5 and PSMB9 are upregulated in resistant cells suggesting a reason for the enhanced proteasomal activity of these cells. The resistant cells showed an altered gene expression profile that indicates a transformation of the parental wild type cell line to acquire resistance. A comparative analysis of the signal transduction pathways operated in these cells showed that many of the activation and cell survival pathways that are present in BCWM.1 cells are inhibited in the resistant cells. For example, BCWM.1 cells show a constitutive activation of AKT and ERK1/2 which are inhibited in the resistant cells thus making them insensitive to the inhibitors of these pathways. Similarly, HSP27 which was earlier shown to contribute to bortezomib induced resistance was completely inhibited in BCWM.1 resistant cells. Interestingly, there is an increase in Bcl-2 protein in BCWM.1/BR cells as compared to WT cells indicating that the resistant cells might be dependent on Bcl-2 family for their survival. Inhibition of Bcl-2 induced potent apoptosis in BCWM.1/BR cells. Thus the results presented here indicate that acquired bortezomib resistance in BCWM.1 cells alters their proteasomal activity, cellular signaling pathways to make them resistant to many of the known therapies but these cells retain the Bcl-2 mediated pathway for targeting thus inhibitors of Bcl-2 may be used in therapy against bortezomib-resistant WM. Disclosures Chanan-Khan: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Immunogen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Resistance to Danusertib (formerly PHA-739358) in BCR-ABL-Positive Cells Is Mediated by Upregulation of the Drug Transporter Abcg2 and Can Be Suppressed in Vitro by Combination Treatment with Imatinib.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1724-1724
Author(s):  
Tim H Brummendorf ◽  
Artur Gontarewicz ◽  
Gunhild Keller ◽  
Jürgen Moll ◽  
Melanie Braig ◽  
...  
Keyword(s):  
Stem Cells ◽  
Combination Therapy ◽  
Board Of Directors ◽  
Research Funding ◽  
Aurora Kinase ◽  
Epigenetic Mechanism ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Resistant Cells

Abstract Abstract 1724 Poster Board I-750 The success of imatinib (IM, formerly STI571, Gleevec®) in the treatment of chronic myeloid leukemia (CML) is compromised by the development of primary or acquired IM-resistance, particularly in advanced phase disease as well as by a limited IM-effect on immature hematopoietic stem cells, emphasizing the need for novel therapeutic strategies. The small molecule inhibitor Danusertib (formerly PHA-739358) potently inhibits Aurora and ABL kinases. Here, the individual contribution of each pathway to the effect of Danusertib was investigated. Starting at very low concentration, a dose-dependent reduction of BCR-ABL activity was observed, whereas inhibition of Aurora kinase activity, assessed by phosphorylation of histone H3-Ser10, required substantially higher concentrations. In primary CD34+ CML cells, including initially quiescent leukemic stem cells, combination therapy with IM and Danusertib revealed a synergistic anti-proliferative activity, which also affected immature CD34+38- cells. Neither mono- nor combination therapy led to substantial induction of apoptosis in quiescent stem cells. Interestingly, under treatment with Danusertib, the emergence of resistant clones in a well-established murine Ba/F3-p210 cell model was considerably less frequent than with IM. Surprisingly, Danusertib-resistant cells did not have mutations in BCR-ABL or Aurora kinase domains and remained IM-sensitive. Analysis of resistance mechanisms using DNA-microarray suggests an overexpression of Abcg2 efflux transporter to be causative for the resistance arising under Danusertib treatment. In support of this finding, stable retroviral overexpression of Abcg2 in parental Ba/F3-p210 cells induced a resistant phenotype against Danusertib. Furthermore, the Abcg2 inhibitor Fumitremorgin C (FTC) could restore the sensitivity of resistant cells to Danusertib. Finally, significant re-expression of Abcg2 in parental Ba/F3-p210 cells upon treatment with the demethylating agent 5-Azacytidine suggests that an epigenetic mechanism might play a role in the regulation of Abcg2 gene expression in resistant clones. Detailed analyses of the methylation patterns of the Abcg2 promoter region are currently being performed. In conclusion, simultaneous in vitro exposure of Ba/F3-p210 cells to Danusertib and IM significantly reduced the emergence of drug resistance, raising hope that both epigenetic modulation of drug transporters involved in development of resistance as well as hypothesis-driven combinations of kinase inhibitors may eventually achieve durable disease control even in 2nd and 3rd line treatment of CML. Disclosures Brummendorf: Bristol Myers Squibb: 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, Patents & Royalties, Research Funding. Moll:Nerviano MS: Employment. Jost:MSD: Research Funding. Bokemeyer:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees. Holyoake:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squib: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Balabanov:Novartis : Research Funding; Bristol Myers Squibb: Research Funding.

scholarly journals Engineered iPSC-Derived NK Cells Expressing Recombinant CD64 for Enhanced ADCC

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 10-11
Author(s):  
Kate Dixon ◽  
Robert Hullsiek ◽  
Kristin Snyder ◽  
Zachary Davis ◽  
Melissa Khaw ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Line ◽  
Board Of Directors ◽  
Research Funding ◽  
Nk Cell ◽  
Target Cells ◽  
Advisory Committees ◽  
High Affinity ◽  
Adcc Assay

Natural killer (NK) cells are innate cytotoxic lymphocytes. They target malignant cells via non-clonotypic receptors to induce natural cytotoxicity and also recognize tumor-bound antibodies to induce antibody-dependent cell-mediated cytotoxicity (ADCC). While ADCC by NK cells is a key mechanism of several clinically successful therapeutic monoclonal antibodies (mAbs), most patients exhibit or acquire resistance to mAb therapies. ADCC by human NK cells is exclusively mediated by the IgG Fc receptor, CD16A (FcγRIIIA). Studies have demonstrated that increasing the binding affinity between CD16A and therapeutic mAbs can augment their clinical efficacy. Given the exquisite specificity and diverse antigen detection of anti-tumor mAbs, we are interested in enhancing the ADCC potency of NK cell-based therapies for various malignancies. CD64 is the only high affinity FcγR family member and binds to the same IgG isotypes as CD16A (IgG1 and IgG3) but with > 30-fold higher affinity. CD64 (FcγRI) is normally expressed by certain myeloid cells but not by NK cells. We generated a recombinant version of this receptor consisting of the extracellular region of CD64 and the transmembrane and intracellular regions of human CD16A, referred to as CD64/16A (figure 1A). An important feature of CD64/16A is that due to its high affinity state, soluble monomeric anti-tumor mAbs can be pre-adsorbed to engineered NK cells expressing the recombinant FcγR, and these pre-absorbed mAbs can be switched or mixed for universal tumor antigen targeting (figure 1B). The engineered NK cells used in our study were derived from genetically edited and clonally derived induced pluripotent stem cells (iPSCs) through a series of stepwise differentiation stages (figure 2). Engineered iPSC-derived NK (iNK) cells can be produced in a uniform and clinically scalable manner (figure 2). In Figure 3, using an in vitro Delfia® ADCC assay, we show that iNK-CD64/16A cells mediated ADCC against SKOV3 cells, an ovarian adenocarcinoma cell line, in the presence of the anti-HER2 therapeutic mAb trastuzumab (Herceptin) or anti-EGFR1 therapeutic mAb cetuximab (Erbitux), when either added to the assay or pre-adsorbed to the iNK cells (figure 3). Considering the high affinity state of CD64, we examined the effects of free IgG in human serum on ADCC by iNK-CD64/16A cells. Using an IncuCyte® Live Cell Analysis System, ADCC was evaluated in the presence or absence of 5% human AB serum, in which free IgG was approximately 50-fold higher than the IgG saturation level of the CD64/16A receptors on iNK cells (data not shown). Despite the high levels of excess free IgG, iNK-CD64/16A cells mediated efficient ADCC when Herceptin was either added to the assay or pre-adsorbed to the cells (figure 4). ADCC assays were also performed with Raji cells, a Burkitt lymphoma cell line, as target cells and the therapeutic mAb rituximab (Rituxan). iNK-CD64/16A cells were added with or without pre-adsorbed Rituxan and the assay was performed in 10% AB serum. Again, iNK-CD64/16A cells mediated effective target cell killing in the presence of serum IgG (figure 5), demonstrating that saturating levels of free IgG did not prevent ADCC. To determine if we can further optimize the function of recombinant CD64, we engineered CD64 with the transmembrane regions of CD16A or NKG2D and signaling/co-signaling domain from CD28, 2B4 (CD244), 4-1BB (CD137), and CD3ζ (figure 6). CD64/16A signals by non-covalent association with the immunoreceptor tyrosine-based activation motif (ITAM)-containing signaling adapters CD3ζ and FcRγ found in the cell membrane, whereas the other recombinant CD64 constructs use ITAM and non-ITAM regions to mediate their signaling. The various recombinant CD64 constructs were initially expressed in NK92 cells (lacks expression of endogenous FcγRs) (figure 7). Using the Delfia® ADCC assay system, we examined the function of each recombinant CD64 construct and found all combinations are able to effectively induce ADCC (figure 8). We are in the process of generating iNK cells with these constructs and testing their ability to kill hematologic and solid tumors in vitro and in vivo. Our goal is to utilize this docking approach to pre-absorb mAbs to iNK cells for adoptive cell therapy. The mAbs would thus provide tumor-targeting elements that could be exchanged as a means of preventing tumor cell escape by selectively and easily altering NK cell specificity for tumor antigens. Figure Disclosures Lee: Fate Therapeutics, Inc.: Current Employment. Chu:Fate Therapeutics: Current Employment. Abujarour:Fate Therapeutics, Inc: Current Employment. Dinella:Fate Therapeutics: Current Employment. Rogers:Fate Therapeutics, Inc: Current Employment. Bjordahl:Fate Therapeutics: Current Employment. Miller:Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding; Nektar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Vycellix: Consultancy; GT Biopharma: Consultancy, Patents & Royalties, Research Funding; Onkimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees. Valamehr:Fate Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company. Walcheck:Fate Therapeutics: Consultancy, Research Funding.

scholarly journals BCL-2, a Therapeutic Target for High Risk Hypodiploid B-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 280-280 ◽  
Author(s):  
Ernesto Diaz-Flores ◽  
Evan Q. Comeaux ◽  
Kailyn Kim ◽  
Kyle Beckman ◽  
Kara L. Davis ◽  
...  
Keyword(s):  
Cell Death ◽  
Acute Lymphoblastic Leukemia ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Advisory Committees ◽  
Chromosomal Number ◽  
Patient Derived Xenograft

Abstract Acute lymphoblastic leukemia (ALL) is the most common cancer of childhood. Specific genetic subsets, including hypodiploid ALL, are associated with particularly high rates of relapse. Despite the poor outcomes of hypodiploid B-ALL with traditional therapeutic approaches, there have been no known effective alternative therapies or novel candidates tested to improve outcome. We hypothesized that new therapeutic targets could by identified by integrated biochemical and genomic profiling, combined with functional drug assays in order to determine which pathways play an essential role in transformation. For biochemical profiling, we analyzed multiple pathways commonly deregulated in leukemias using phosphoflowcytometry (including receptor tyrosine kinases, JAK/STAT, MAPK, PI3K, PTEN, Bcl-2 survival and pro-apoptotic family members and p53). We subjected hypodiploid cell lines (NALM-16, MHH-CALL2) and patient derived xenograft samples in vitro to inhibitors against each of these pathways (PP2:Src family;Ruxolitinib: JAK/STAT; PD235901/CI1040: MAPK; GDC-0941, PI-90, PI-103, p110 (a, b, g, d): PI3K isoform specific; PP-242:mTOR; ABT-263/ABT-737: Bcl-2/Bcl-xl, and ABT-199: Bcl-2 specific). We found that the Bcl-2 inhibitors (ABT-263, ABT-737 and ABT-199) and to a lesser extent PI3K pathway inhibitors GDC-0941 and PP-242, but not the MAPK or RTK inhibitors, efficiently reduced proliferation of hypodiploid cells. However, only ABT-263/ABT-199 induced high levels of apoptosis at nanomolar concentrations. Based on the consistent efficacy observed with ABT-199 against hypodiploid patient-derived cells and cell lines in culture, we selected eight cryopreserved, previously xenografted (F3 generation) hypodiploid patient samples (4 low hypodiploid, chromosomal number between 32 and 39; and 4 Near Haploid, chromosomal number between 24 and 31) and three non-hypodiploid patient samples (Ph-positive,Ph-Like and Erg+) for a preclinical trial in immunodeficient mice. Each patient sample was engrafted into six mice, which were randomized to receive vehicle or ABT-199 daily over 60 days (Figure 1). Treatment started when the peripheral blood (PB) human CD45 count reached 15%. A rapid decrease in PB blasts was noted at 7 days (Figure 1). Eighty-five percent of the hypodiploid xenografts survived 60 days with either undetectable or low levels of leukemia in the PB. In contrastPh+ andPh-Like xenografts died within 10-20 days regardless of treatment. Importantly, hypodiploid leukemic blasts gradually emerged after discontinuing ABT-199 after 60 days. Additionally, despite low or undetectable levels of leukemic blasts in PB and reduced levels in bone marrow and spleen, all mice had high percentages of leukemic cells in the liver (Figure 2). In conclusion we have identified the survival protein Bcl-2 as a promising molecular target in hypodiploid B-ALL. ABT-199 for dramatically reduced leukemia cells in vitro and in vivo in patient-derived xenograft models of hypodiploid B-ALL. However, the liver represented a protective niche for these leukemias. In addition, our biochemical characterization of the organ infiltrating blasts collected from mice on trial indicate that the sensitivity of hypodiploid ALL to ABT-199 relies not only on high levels of Bcl-2 and deficiency for other survival proteins such as Bcl-xl but also on high levels of proapoptotic proteins, providing two different signatures that correlate with response to ABT-199. Using genome editing (CRISPR/Cas9) we interrogated the necessity for individual proapoptotic genes, including PUMA, NOXA, and BAD, for ABT-199-induced cell death. This study provides encouraging preclinical data that Bcl-2 may be a promising target for the treatment of hypodiploid B-ALL. Our studies identify signature biomarkers that correlate with drug response and identify essential proteins mediating ABT-199-induced cell death. Importantly, this report also identifies the limitations of using ABT-199 as single drug, and provides the rationale for using combinatorial therapies in order to improve the efficacy of the drug. Disclosures Mullighan: Loxo Oncology: Research Funding; Amgen: Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees. Loh:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding.

Novel Selective Orally Bioavailable Small Molecule PAK4 Allosteric Modulators (PAMs) Display Anti-Tumor Activity in Vitro and in Vivo in Hematological Malignancies

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2208-2208
Author(s):  
William Senapedis ◽  
Ryan George ◽  
Dilara McCauley ◽  
Joel Ellis ◽  
Marsha Crochiere ◽  
...  
Keyword(s):  
Cell Line ◽  
Board Of Directors ◽  
Small Molecule ◽  
Allosteric Modulators ◽  
Advisory Committees ◽  
Hematological Cancers ◽  
Orally Bioavailable ◽  
Anti Tumor Activity

Abstract Introduction: Many hematological cancers have been successfully treated through identification of specialized targets in each specific tumor subtype (e.g. BTK inhibition in NHL or proteasome inhibition in multiple myeloma). The p21-Activated Kinase 4 (PAK4) is critical to cellular signaling and may represent a new target for therapy in many hematologic malignancies. PAK4 is a member of the PAK family of proteins that regulate cell survival, cell division and apoptosis. The six members of the PAK family are divided into two groups; Group I (PAK1, 2, 3) and Group II (PAK4, 5, 6), based upon their sequence homology and regulatory mechanisms. PAK4 is a member of the group II family of PAKs and is amplified or mutated in many cancer types. PAK4 is also a key downstream effector of the K-Ras pathway. Methods: Flow cytometry and CellTiter AQueous One (MTS) assays were used to determine compound effects on cell cycle distribution, proliferation and viability. Immunoblots were used to measure effects of compounds on protein steady state levels and phosphorylation. The T-cell ALL cell line, MOLT-4, and the mantle cell lymphoma cell line, Z-138, were used in xenograft models in mice to test the in vivo efficacy of these compounds. Results: We have identified selective, orally bioavailable, small molecule PAK4 allosteric modulators {PAMs; e.g. KPT-8752 (mw: 585.6), KPT-9274 (mw: 610.6), and KPT-9331 (mw: 628.6)} which demonstrated selective anti-tumor activity in a variety of hematological cancer cell lines (IC50 values = 0.005 – 1 mM). Treatment of cancer cells with these small molecules resulted in the reduction of PAK4 steady state levels and reduced phosphorylation of key growth signaling proteins such as Akt, β-catenin, cofilin, p21, and cyclin D1. There was a measurable increase in phospho-AMPK indicative of autophagy and stress. These allosteric modulators induced apoptosis through the activation of caspases 3 and 8 and subsequent cleavage of PARP. In MOLT-4 and Z-138 xenograft mouse models, daily treatment with oral PAMs resulted in near elimination of small (100 mm3) and large (800 mm3) tumors in the absence of any clinical signs of toxicity within the animals. Additional cell line and primary tumor models are currently being explored. Conclusions: PAK4 represents a novel anti-cancer target as a major downstream effector of the Ras oncogene. We have identified selective, orally-bioavailable small molecule PAK4 allosteric modulators which induce potent cytotoxicity in multiple leukemia and lymphoma cell lines with minimal toxicity to normal cell in vitro and clear anti-tumor activity with excellent tolerability in in vivo models of hematological cancers. These compounds inactivate PAK4 by directly inducing PAK4 destabilization. This represents a novel mechanism of the protein kinase inactivation involving degradation of PAK4 rather than direct inhibition of the kinase activity. Based on the in vitro and in vivo activity, these PAK4 allosteric modulators show promising results for the treatment of a wide variety of hematological cancers. Disclosures Senapedis: Karyopharm: Employment. George:Karyopharm: Employment. McCauley:Karyopharm Therapeutics: Employment, Equity Ownership. Ellis:Karyopharm: Employment. Crochiere:Karyopharm: Employment. Savona:Karyopharm: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees. Shacham:Karyopharm Therapeutics: Employment. Landesman:Karyopharm Therapeutics: Employment. Baloglu:Karyopharm: Employment.

Indirubins: A Potential Therapeutic Target in Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3259-3259
Author(s):  
Tina Bagratuni ◽  
Nicolas Gaboriad-Kolar ◽  
Roubini Zakopoulou ◽  
Vassilios Myrianthopoulos ◽  
Efstathios Kastritis ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Death ◽  
Virtual Screening ◽  
Cell Line ◽  
Board Of Directors ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Inhibitory Effects ◽  
Advisory Committees

Abstract Current drugs in the treatment of Multiple Myeloma (MM) result in cell death via a number of mechanisms including a direct effect on plasma cells as well as alteration in the BM microenvironment. Although effective to some extent, none of the drug mechanisms of action are fully targeting a biological process essential and necessary for PC survival. Among the FDA approved kinase inhibitors, few are based on natural scaffolds. 6-bromoindirubin-3'-oxime (6BIO) is a potent kinase inhibitor based on the natural 6-bromoindirubin scaffold. Indirubin and 6-bromoindirubin are two natural products that have found a particular interest in dye chemistry as the main constituent of indigo and Tyrian purple dyes. Recent findings discovered that 6BIO was a promising anti-cancer agent acting on the JAK/STAT signaling pathway mediating cell proliferation. After enhancement of the chemical structure of 6BIO, further reports exposed that MLS-2438 and MLS-2384 were Akt signaling pathway inhibitor (MLS-2438) and potent c-Src kinase direct inhibitors. A library containing 2000 natural molecules was constructed using several data platforms. Each molecule was processed through different filters such as tautomeric studies, protonation and steroisomerism status in order to be used for calculations of virtual evaluation. Two approaches were followed: the structure-based virtual screening and the ligand-based virtual screening. To achieve structural based virtual screening, binding and evaluation of the chemical relation of each molecule in the crystallographic structure of the proteasome β5 subunit was performed. In the ligand-based virtual screening, calculations were made to identify the structural similarities of each molecule with the known proteasome inhibitor, bortezomib. The results of both approaches were combined, the molecules ranked, and 100 out of 2000 were identified as strong potential bioactive hits for the β5 subunit. Out of these 100 molecules, the chemical structures of high interest were the following: indole alkaloids derivatives (indirubins), flavonoids, secoiridoids, simple phenolic acids and acetophenone. A rational selection of indirubins derivatives was conducted in order to study their cytotoxic effects on MM. Fifty indirubins derivatives were selected based on different criteria: structure, known/unknown targets, chemodiversity in substitution patterns. To explore the inhibitory effects of indirubins in MM, we performed the WST1 proliferation assay in three MM cell lines (H929, JJN3, L363). Initially, all the selected indirubins (~50 indirubins) were tested at 7.5μM in L363 cell line and proliferation results from the WST1 assay extracted after 24 hours of treatment. More than half of the indirubins tested displayed more than 50% reduction of the proliferation at 7.5μM. Interestingly, 10 out of the 50 indirubins tested reduced more than 80% proliferation after 24 hours. The most active indirubins were tested in H929 and JJN3 cell lines, where similar effects were seen after 24 hours of treatment. All tested indirubins acted in a dose-dependent manner. Based on our first set of data, we suggest that indirubins have significant anti-proliferative effects on MM cell lines. Among the most active indirubins, two molecules namely 805 and 673 emerged as attractive for further development. Compound 805 is an analog of MLS-2384 while compound 673 is an analog of MLS-2438. The latter derivative represents a promising candidate displaying an IC50below the micromolar range on H929 and JJN3 cells. To determine the kind of cell death caused by one of the most active indirubins, 673, cell cycle analysis was performed before and after treatment in H929 cell line. In particular changes in RNA expression of 84 genes key to cell cycle regulation were analyzed in H929 cell line. Our results show that among other genes, the ones which have a dramatic increase in their expression (>5 fold) are mainly involved in cell cycle arrest such as GADD45A, CDC34, TP53, CHEK1 and CHEK2. A more detailed analysis of the profiler array will be presented at the meeting. In conclusion, this is the first study to show the inhibitory effects of indirubins in MM. Further investigation of these compounds may offer a therapeutic advantage that would affect MM pathogenesis and treatment. Disclosures Kastritis: Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Genesis: Consultancy, Honoraria. Terpos:Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Dimopoulos:Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genesis: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Dual Targeting of Ph+ ALL with Dasatinib and ABT-199 (Venetoclax)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1329-1329
Author(s):  
Jessica Leonard ◽  
Joelle Rowley ◽  
Brandon Hayes-Lattin ◽  
Jeffrey W. Tyner ◽  
Marc Loriaux ◽  
...  
Keyword(s):  
Older Adults ◽  
Cell Line ◽  
Board Of Directors ◽  
Research Funding ◽  
Mononuclear Cells ◽  
Annexin V ◽  
Advisory Committees ◽  
Equity Ownership

Abstract Introduction: Treatment of adult Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL) remains a challenge. While the addition of the targeted tyrosine kinase inhibitors (TKI) to standard cytotoxic therapy has greatly improved upfront treatment, treatment related mortality in older adults remains high. A novel induction regimen combines the targeted dual Abl/Src TKI Dasatinib (Sprycel, BMS) with a corticosteroid. After the first 21 days of induction the corticosteroids are tapered due to significant toxicities, particularly in older adults. Unfortunately, remaining on TKI monotherapy renders patients susceptible to the development of TKI resistance and thus identifying targeted agents that could enhance the activity of TKIs is urgently needed. Recently a novel and selective inhibitor of BCL-2, ABT-199 (Venetoclax, AbbVie) has shown impressive activity against other lymphoid malignancies including CLL and NHL. Here we describe the pre-clinical and in vivo efficacy of ABT-199 in combination with dasatinib in Ph+ ALL and propose its potential use in future clinical trials. Methods: Drug efficacy in vitro was determined using the Ph+ ALL cell line SupB15, primary Ph+ ALL sample (12-149), the dasatinib sensitive Pre-B ALL cell line RCH and the CML cell line K562. Cells were treated with dasatinib, ABT199 or in combination for 72 hours. Cell viability was assessed with the colorimetric MTS assay and apoptosis was assessed with annexin V staining. Expression of the BCL family proteins BCL-2 and MCL-1 were assessed via immunoblot. Immunodeficient NSG mice were injected with 12-149, then one week later treated with vehicle, 5 mg/kg dasatinib, 5 mg/kg ABT-199, or the combination daily for 5 days each week. Peripheral blood was obtained every 1-2 weeks to assess for engraftment as defined by the presence of >10% human CD45+ cells in the peripheral blood. Once engrafted, mice were euthanized and examined. Mononuclear cells were extracted and assessed for BCL2 and MCL1 expression. Statistical methods were performed using Calcusyn and PRISM. Results: Susceptibility to BCL2 inhibition: Of the dasatinib sensitive cells tested, SupB15 and 12-149 cells were susceptible to ABT-199 while RCH and K562 cells were not. The ALL cells expressed BCL-2 while the CML cell line expressed BCLx. SupB15 expressed low levels of the antiapoptotic protein MCL1 while RCH cells had relatively higher levels. siRNA of MCL-1 rendered the RCH cells sensitive to inhibition by ABT-199. In SupB15 cells, treatment with ABT-199 alone led to upregulation of MCL-1 at 24h which was prevented by the combination of dasatinib + ABT199. Synergy in Ph+ ALL: The calculated IC50 of dasatinib and ABT199 in SupB15 were 8.8nM and 5.9nM, respectively. The IC50 of equimolar combination was 0.42nM, and synergistic with combination index (CI) values between 0.15 and 0.49. Primary Ph+ ALL xenograft cells showed a similar pattern of synergy to the dasatinib + ABT199 combination. Combination treatment also greatly increased apoptosis as measured by Annexin V staining. Xenograft Studies: Animals were treated with a ten-fold lower dose of dasatinib and ABT199 from prior published data. There was no significant difference in time to engraftment or disease burden between vehicle or single agent ABT-199. In contrast, less than one half of the animals treated with dasatinib engrafted by 90 days while none of the animals treated with both dasatinib and ABT-199 engrafted. Most intriguing was the decrease in disease burden as measured by splenic size in the combination group compared to all other groups (P<0.0001, one-way ANOVA). Analysis of BCL-2 family proteins from mononuclear cells isolated from untreated animals confirmed upregulation of BCL-2 and relatively low levels of MCL-1. Animals treated with ABT-199 had greatly upregulated levels of MCL-1, while those treated with dasatinib or the combination did not. Conclusions: The combination of ABT-199 with dasatinib synergistically targets Ph+ ALL cells both in vitro and in vivo, laying the foundation for further evaluation in vivo for adult Ph+ ALL. As demonstrated by others, malignancies that are particularly susceptible to BCL targeting are those which display high BCL-2 expression and a low MCL-1: BCL-2 ratio. Combined targeted therapies may offer the potential for greater and longer responses without the morbidity associated with cytotoxic chemotherapy, particularly in older adults. Disclosures Tyner: Aptose Biosciences: Research Funding; Janssen Pharmaceuticals: Research Funding; Incyte: Research Funding; Array Biopharma: Research Funding; Constellation Pharmaceuticals: Research Funding. Druker:Cylene Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oregon Health & Science University: Patents & Royalties; McGraw Hill: Patents & Royalties; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Aptose Therapeutics, Inc (formerly Lorus): Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Millipore: Patents & Royalties; Fred Hutchinson Cancer Research Center: Research Funding; Novartis Pharmaceuticals: Research Funding; Sage Bionetworks: Research Funding; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; ARIAD: Research Funding; Henry Stewart Talks: Patents & Royalties; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncotide Pharmaceuticals: Research Funding; CTI Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding; Roche TCRC, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Consultancy.

scholarly journals Preclinical Activity of Novel MCL1 Inhibitor AZD5991 in Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 952-952 ◽  
Author(s):  
Shannon M Matulis ◽  
Vikas A. Gupta ◽  
Izabelle Brown ◽  
Jonathan J Keats ◽  
Paul Secrist ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Line ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Contact ◽  
Advisory Committees ◽  
Highly Sensitive ◽  
Ic50 Values ◽  
Cell Cell

Abstract We and others have previously demonstrated that MM is often dependent on MCL1 or co-dependent on MCL1 and BCLXL or BCL2 for survival. Therefore, drug development targeting MCL1 has been a top priority. Here we report on AZD5991, a specific small molecule inhibitor of MCL1. We treated 17 MM cell lines with increasing concentrations of AZD5991 for 24 h and measured Annexin V staining to determine the IC50s. Nine of the cell lines tested were highly sensitive to AZD5991 with IC50 values below 100 nM, 6 lines exhibited an intermediate sensitivity (IC50 100-1000 nM), and only 2 cell lines tested were resistant (IC50 >1000 nM). Six of the highly sensitive lines are t(11;14) and sensitive to venetoclax suggesting co-dependence on BCL2 and MCL1 for survival. We also determined the effect of the bone marrow microenvironment on the response of MM cell lines to AZD5991. We reported that IL-6 protects MM cell lines and patient samples from apoptosis by making the cells more MCL1 dependent. Based on this, we predicted IL-6 would have little to no effect on AZD5991-induced cell death. We treated 12 cell lines with AZD5991 in the presence of 1 ng/mL IL-6 or 10% Hs5 conditioned medium (CM) for 24 h and found that only 3/12 and 2/12 lines were protected from apoptosis in the presence of IL-6 and CM, respectively. Interestingly, when co-cultured with the stromal cell line Hs5, 7/11 lines tested were protected from AZD5991-induced cell death, suggesting cell-cell contact is influencing the response. This is in contrast to ABT-737 and venetoclax where cell-cell contact provided no additional protection than CM. Mechanistically apoptosis induced via MCL1 inhibition is not dependent on BIM expression as is the case with BCLXL and BCL2 inhibition. KMS26 and LP1 MM cell lines contain a bi-allelic deletion of BIM and we have reported their resistance to ABT-737. However, both cell lines respond to AZD5991 with IC50 values in an intermediate sensitivity range. Co-immunoprecipitation (CoIP) studies were employed to determine the protein bound to MCL1 that could be promoting apoptosis upon release. We found NOXA and BAK bound in KMS26 and LP1 and both were released from MCL1 in response to AZD5991. Additionally, CoIPs performed on cell lines expressing BIM showed NOXA, BIM, and BAK bound to MCL1 and released following treatment. To further investigate we used CRISPR-cas9 to generate MM cell lines lacking expression of NOXA, BAK, BAX, or BIM. In KMS26 and LP1, deletion of NOXA and BAX had little effect on AZD5991-induced cell death while the BAK deletion significantly inhibited apoptosis in both cell lines. Similar results were observed in the BIM expressing cell line OCI-My5, with no protection from AZD5991-induced apoptosis in the NOXA and BAX edited lines, significant protection in the BAK-deleted line, and an intermediate degree of protection in the BIM knockout line. In KMS18, BIM deficiency had a minimal effect on apoptosis following MCL1 inhibition, however both BAX and BAK were required for AZD-induced cell death. Additionally, we have tested 41 samples from 37 patients for sensitivity to AZD5991. Samples were treated with increasing concentrations to determine IC50 values in the same manner as the MM cell lines. The samples segregated into 4 groups based on IC50. The most sensitive group (N=3) had an IC50 below 10 nM. The largest group had an IC50 range of 50-114 nM (N=26). The last two cohorts were more resistant with a range of 500-916 nM (N=10) and 2 samples with an IC50 over 1300 nM. Since MCL1 is on 1q21, a frequently amplified region in MM, we determined if 1q21 gain was associated with sensitivity. For the 35 samples where FISH data were available, 18 had 1q21 gains by FISH while 17 were negative. There is a trend for the 1q21 gain cohort to be more sensitive (P=0.0573), with only 2/18 having an IC50 above 109 nM. In contrast for the 1q21 negative 7/17 were in the resistant groups. Thus 1q21 may be a marker of sensitivity to MCL1 inhibitors. The data reported here demonstrate that AZD5991 is effective at inducing apoptosis in MM and can overcome soluble microenvironment resistance factors that influence the response to venetoclax. This appears to be due to differential requirements for pro-apoptotic factors for BCL2 and MCL1 inhibition and suggests an underappreciated complexity in the role of BCL2 and MCL1 in cell survival. Finally these findings also suggest that 1q21 gain may be a marker for AZD5991 sensitivity. A clinical trial is currently ongoing in myeloma. Disclosures Secrist: AstraZeneca: Employment. Cidado:AstraZeneca: Employment, Equity Ownership. Tron:AstraZeneca: Employment. Neri:Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Bahlis:Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding. Kaufman:Roche: Consultancy; Abbvie: Consultancy; Karyopharm: Other: data monitoring committee; Janssen: Consultancy; BMS: Consultancy. Heffner:Pharmacyclics: Research Funding; Genentech: Research Funding; ADC Therapeutics: Research Funding; Kite Pharma: Research Funding. Lonial:Amgen: Research Funding. Nooka:Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Adaptive technologies: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Spectrum Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Boise:AstraZeneca: Honoraria; Abbvie: Consultancy.

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