Potent Ikaros Degradation By the Cereblon E3 Ligase Modulator CC-92480 Is Effective in Combination with Menin-MLL1 Inhibition in MLL1-Rearranged and NPM1-Mutant AML

Abstract Acute myeloid leukemia (AML) has a poor prognosis despite intensive therapy. Novel therapies directed at molecular drivers of AML are needed. Ongoing clinical trials with inhibitors of the Menin and Mixed Lineage Leukemia 1 (KMT2A/MLL1) protein-protein interaction for patients with MLL1-rearranged (MLL1-r) and Nucleophosmin mutant (NPM1c) AML have promising early results. We have identified Ikaros degradation as a synergistic therapeutic target with Menin-MLL1 inhibition in MLL1-r AML and identified the novel cereblon E3 ligase modulator (CELMoD) CC-92480 as an efficacious compound in vitro and in vivo in MLL1-r and NPM1c AML models. To find genetic targets that synergize with Menin-MLL1 inhibition, we performed a genome-scale CRISPR-Cas9 functional genetic screen in the Molm-13 (MLL1-AF9) AML cell line. Using the MAGeCKFLUTE pipeline, comparison of 14 days of treatment with VTP-50469 (Menin-MLL1 inhibitor) versus DMSO identified IKZF1 deletion as one of the most negatively selected genes when treated with VTP-50469 (IKZF1 codes for the hematopoietic transcription factor Ikaros). To validate this finding, CRISPR-Cas9-based competition assays were performed in Cas9-expressing MLL1-r and NPM1c AML cell lines by monitoring of sgRNA-RFP expression over time via flow cytometry. IKZF1 was a dependency in all four MLL1-r cell lines and both NPM1c cell lines evaluated. The sgRNAs targeting IKZF1 depleted faster when cells were treated with VTP-50469. To evaluate the effect of Ikaros protein degradation in these AML cell lines, we assessed proliferation upon treatment with three Ikaros-degrading compounds: the Immunomodulatory Imide Drug (IMiD) lenalidomide and the CELMoDs CC-220 and CC-92480. CELMoDs are a derivative class of IMiDs with much more potent Ikaros degradation (lenalidomide < CC-220 < CC-92480) and Phase I activity in multiple myeloma patients refractory to prior IMiD therapy. Cells were plated with 5 different doses per drug. Every 3 days cells were split, drug was replenished, and live cell counts were obtained using viability staining and flow cytometry. Evaluation of the absolute IC 50 in 7 MLL1-r or NPM1c AML cell lines at day 9 showed that 6 cell lines were sensitive to CC-92480 (absolute IC 50 range: <0.1-to-3.6 nM), 5 were sensitive to CC-220 (absolute IC 50 range: 15.7-to-215.3 nM), and only two were sensitive to lenalidomide (absolute IC 50 of 267.0 nM and 555.3 nM). These data suggest that greater Ikaros degradation leads to greater efficacy in vitro. Lenalidomide, CC-220, and CC-92480 all synergized with VTP-50469 to inhibit proliferation. Apoptosis assays were performed in the MLL1-r AML cell lines Molm-13 and MV4;11 using annexin V staining following 6 days of treatment with DMSO, lenalidomide, CC-220, and CC-92480 with and without VTP-50469. For both cell lines, 5 µM lenalidomide, 1 µM CC-220, and 3 nM CC-92480 increased apoptosis less than two-fold as single agents while VTP-50469 did so only 2.5-fold and 3.7-fold compared to DMSO control. Strikingly, the combination of each of these IMiD or CELMoD doses with VTP-50469 induced apoptotic markers at least 7.5-fold compared to DMSO control. This cooperativity between Ikaros protein degradation and Menin-MLL1 inhibition could be explained by their combined targeting of a HOXA/MEIS1 transcriptional program as Ikaros degradation in MLL1-r AML cell lines perturbs expression of HOXA9 target genes without altering the expression of HOXA9 itself. We next tested CC-92480 in four patient derived xenograft (PDX) models of AML: one NPM1c and three MLL1-r models. In these experiments, NSG-S mice were engrafted with PDX samples (>7 mice per treatment cohort). Following detection of leukemia in peripheral blood, mice were randomized to receive vehicle control, lenalidomide 50 mg/kg (in two of the four PDXs), or CC-92480 10 mg/kg for 28-55 days and survival benefit was assessed. In these models, CC-92480 increased median survival compared to DMSO by 28.3% (p < 0.05), 66% (p <0.0005), 128% (p < 0.0005), and 133% (p < 0.0001). In two of these experiments CC-92480 was also compared to lenalidomide and increased survival by 23.4% (p < 0.0005) and 28.1% (p < 0.05). In summary, the preclinical activity of CC-92480 in MLL1-r and NPM1c AML models, particularly in combination with Menin-MLL1 inhibition, supports translation of this compound or a similarly potent, Ikaros-degrading CELMoD into clinical trials for these molecular subtypes of AML. Disclosures Aubrey: Walter and Eliza Hall Institute of Medical Research: Patents & Royalties: Receives proceeds from Royalties and Milestone payments related to the BCL2-inhibitor, ABT-199/venetoclax. McGeehan: Syndax Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Fischer: Neomorph Inc.: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; C4 Therapeutics: Current equity holder in publicly-traded company; EcoR1 Capital: Consultancy; Sanofi: Consultancy; Astellas: Consultancy; Deerfield: Consultancy; RA Capital: Consultancy; Novartis: Research Funding; Astellas: Research Funding; Deerfield: Research Funding; Ajax: Research Funding; Jengu Therapeutics: Current holder of individual stocks in a privately-held company; Civetta Therapeutics: Current holder of individual stocks in a privately-held company. Armstrong: AstraZeneca: Research Funding; Syndax: Research Funding; Novartis: Research Funding; Janssen: Research Funding; Mana Therapeutics: Consultancy; Accent Therapeutics: Consultancy; OxStem Oncology: Consultancy; C4 Therapeutics: Consultancy; Cyteir Therapeutics: Consultancy; Vitae/Allergan Pharma: Consultancy; Imago Biosciences: Consultancy; Neomorph Inc: Consultancy, Current holder of individual stocks in a privately-held company.

Download Full-text

Preclinical Activity of the Clinical Stage Protein Arginine Methyltransferase 5 (PRMT5) Inhibitor PRT543 in Splicing Mutant Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML)

Blood ◽

10.1182/blood-2021-148494 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2597-2597

Author(s):

Leya Y Schwartz ◽

Gaurav S Choudhary ◽

Nandini Ramachandra ◽

Srabani Sahu ◽

Shanisha Gordon ◽

...

Keyword(s):

Flow Cytometry ◽

Research Funding ◽

Clinical Stage ◽

Interleukin 1 ◽

Splicing Factor ◽

Myeloid Differentiation ◽

Large Set ◽

Publicly Traded ◽

Exon 4

Abstract MDS and AML are generally incurable malignancies that need newer therapeutic options, as the disease-initiating stem cells are not eliminated by conventional therapies. Splicing factor mutations account for approximately 50% of mutations in MDS. Among those are SF3B1 and U2AF1 mutations, which are related to pathogenesis of disease by overactivation of oncogenic pathways, such as Interleukin-1 Receptor-Associated Kinase 4 (IRAK4) signaling. Via activation of IRAK4 and other pathways, spliceosome mutations can lead to a block in differentiation and malignant proliferation. PRMT5 is an enzyme involved in spliceosome complex formation and fidelity and is over-expressed in patients with MDS/AML. The inhibition of PRMT5 may contribute to stem/progenitor cell differentiation rather than aberrant proliferation in an undifferentiated immature state. The objective of this study is to determine the activity of a clinical stage PRMT5 inhibitor, PRT543, in subtypes of MDS/AML using cell lines and primary samples. In preclinical studies PRT543 showed broad antitumor activity in vitro and in vivo (Bhagwat AACR 2020) and is currently under investigation in a Phase I clinical trial in patients with myeloid malignancies. We used a K562 cell line with CRISPR-introduced SF3B1 K700E mutation and isogenic control (K700K) in proliferation and myeloid differentiation assays with PRT543. The SF3B1 K700E mutant cells showed myeloid differentiation after treatment with the PRT543 PRMT5 inhibitor, as assessed by single cell colony assays and flow cytometry, while no substantial effects were observed in controls (K700K). We next evaluated PRMT5 expression in a large set of MDS CD34+ cells and observed substantial overexpression in SF3B1 mutant samples. Primary MDS/AML progenitors were cultured in methylcellulose colony forming unit (CFU) assays and treated with PRT543 at multiple concentrations versus vehicle controls. A majority of the SF3B1 patient samples showed a substantial increase in erythroid differentiation as assessed by colony formation and flow cytometry in the presence of 1nM and 5nM PRT543. In non-SF3B1 mutated patient samples, there was no clear difference in differentiation in the presence of PRT543. We next evaluated whether PRMT5 inhibition led to inhibition of oncogenic IRAK4 pathways. Retention of exon 4 of IRAK4 occurs in splicing mutant MDS and leads to production of an active long IRAK4 isoform. As measured by RNA-seq, PRMT5 inhibition led to decreased retention of exon 4 in IRAK4 transcripts. This decrease in the IRAK4 long form in response to PRT543 treatment was confirmed by immunoblotting, demonstrating reduction of this oncogenic signaling pathway. In summary, PRMT5 inhibition with PRT543 can release a differentiation block in MDS/AML, specifically in splicing mutant samples. PRMT5 inhibition decreases IRAK4-long isoform expression providing a potential mechanism for its activity in splicing factor mutant cases. Disclosures Ruggeri: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Heiser: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Scherle: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Starczynowski: kurome Inc: Consultancy. Verma: Throws Exception: Current equity holder in publicly-traded company; Stelexis: Current equity holder in publicly-traded company; Celgene: Consultancy; Acceleron: Consultancy; Novartis: Consultancy; Stelexis: Consultancy, Current equity holder in publicly-traded company; Eli Lilly: Research Funding; Curis: Research Funding; Medpacto: Research Funding; Incyte: Research Funding; GSK: Research Funding; BMS: Research Funding.

Download Full-text

AT-101, a Small Molecule Inhibitor of Bcl-2 Family Proteins, Has Significant In Vitro Activity in Multiple Myeloma.

Blood ◽

10.1182/blood.v106.11.1581.1581 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1581-1581

Author(s):

Shaji Kumar ◽

Michael Kline ◽

Terry Kimlinger ◽

Michael Timm ◽

Jessica Haug ◽

...

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

Clinical Trials ◽

Cell Lines ◽

Myeloma Cell ◽

Time Dependent ◽

Myeloma Cells ◽

Molecule Inhibitor ◽

Apoptotic Proteins

Abstract Background: Multiple myeloma (MM) is a plasma cell proliferative disorder that results in considerable morbidity and mortality. As it is incurable with the current therapeutic approaches, more effective therapies based on better understanding of the pathobiology of the disease are needed. In MM, malignant plasma cells are characterized by low proliferative and apoptotic rates compared to other malignancies. Studies have shown elevated expression of anti-apoptotic proteins of the Bcl-2 family in MM cells, which appear to correlate with resistance to therapy with certain drugs. Hence, accelerating the apoptotic process by targeting the Bcl-2 family of proteins appears to be an attractive strategy for the treatment of MM. AT-101 is an orally bioavailable derivative of gossypol in cancer clinical trials, and is being developed by Ascenta Therapeutics. AT-101 behaves as a small molecule inhibitor of Bcl-2 and Bcl-XL, binding to the BH3-binding pocket of these proteins and inhibiting their ability to suppress the activity of pro-apoptotic proteins, resulting in apoptosis. Methods and Results: AT-101 was cytotoxic to several different myeloma cell lines with a median effect observed at around 5μM concentration using an MTT cell proliferation assay. Additionally, at similar doses AT-101 induced cytotoxicity in myeloma cell lines resistant to conventional agents such as Melphalan (LR50), Doxorubicin (Dox40) and Dexamethasone (MM1.R), indicating non-overlapping mechanisms. To evaluate the ability of the drug to induce cell death in the tumor microenvironment, MM cells were co-cultured with marrow stromal cells or in the presence of VEGF or IL-6, two cytokines known to be important for myeloma growth and survival. AT-101 was cytotoxic to myeloma cells under these conditions as well with a median effect at concentrations of 5–10μM. AT-101 was able to induce apoptosis in myeloma cells in a dose- and time dependent fashion, as demonstrated by flow cytometry using Annexin/PI staining as well as cell cycle studies. AT-101 also resulted in cytotoxicity of freshly isolated primary patient myeloma cells, inducing apoptosis in a dose dependent manner. We also studied the effect of AT-101 on levels of different pro- and anti-apoptotic proteins using flow cytometry on permeabilized cells. A time-dependent increase in the level of BAX was observed following treatment with AT-101 without any associated change in levels of Bcl-xL or Bcl-2. Further studies evaluating the combination of AT101 with other active myeloma agents as well as a detailed evaluation of its mechanisms in myeloma are ongoing. Conclusion: AT-101 has significant activity in vitro in the setting of myeloma as demonstrated by its effect on myeloma cell lines and primary patient cells. More importantly, it has activity against cell lines resistant to conventional anti-myeloma agents. In addition, Phase I studies with this agent are currently ongoing in patients with solid tumors. The results from these studies form the rationale for early phase clinical trials in MM, either alone or in combination with other active therapies.

Download Full-text

Significant in-Vitro Activity of a Novel JAK2 Inhibitor in Multiple Myeloma Associated with Preferential Cytotoxicity for CD45+ Myeloma Cells.

Blood ◽

10.1182/blood.v114.22.2848.2848 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2848-2848

Author(s):

Vijay Ramakrishnan ◽

Jessica Haug ◽

Teresa Kimlinger ◽

Timothy Halling ◽

Linda Wellik ◽

...

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

Cell Lines ◽

Research Funding ◽

Myeloma Cell ◽

Vitro Activity ◽

In Vitro Activity ◽

Myeloma Cells ◽

Stat Pathway

Abstract Abstract 2848 Poster Board II-824 Background: Multiple myeloma remains incurable with current therapies and novel approaches based on disease biology are needed. IL-6 is a critical cytokine involved in myeloma cell proliferation and survival and exerts its activity primarily through the JAK/STAT pathway. In addition to IL6, other cytokines are also believed to cross talk with the JAK/STAT pathway, making it a crucial interface for survival signals. It has been implicated in myeloma cell interaction with the microenvironment and resistance to apoptotic stimuli from different drugs, and represents a potential therapeutic target. We examined the pre-clinical activity of a novel JAK2 tyrosine kinase inhibitor TG101209. Methods: TG101209 (N-tert-butyl-3-(5-methyl-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-pyrimidin-4-ylamino)-benzenesulfonamide) was synthesized by TargeGen Inc. (San Diego, CA, USA). Stock solutions were made in DMSO, and subsequently diluted in RPMI-1640 medium for use. MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum (20% serum for primary patient cells) supplemented with L-Glutamine, penicillin, and streptomycin. Cytotoxicity was measured using the MTT viability assay and proliferation using thymidine uptake. Apoptosis was measured using flow cytometry upon cell staining with Annexin V-FITC and propidium iodide (PI) for cell lines and using Apo2.7 in primary patient cells. CD45 expression was estimated using flow cytometry and cells were gated by their CD45 expression to assess differential effects of the drug. Immunoblotting was done on cell extracts at various time points following incubation with the drug in order to study the cell signaling pathways. Results: TG101209 resulted in a dose and time dependent inhibition of cell growth in the MM cell lines tested. Most of the cytotoxicity was evident by 48 hours, with minimal increase seen up to 96 hours of incubation. At 48 hours of incubation, the median inhibitory concentration was between 2 and 4uM with similar IC50 seen for myeloma cell lines sensitive or resistant to conventional therapies. The IC50s were maintained when the cells were treated in co-culture with stromal cells or in the presence of IL6, IGF or VEGF. Increasing doses of IL6 was not able to rescue the cells from the drug. Dose dependent decrease in proliferation of the cell lines was evidenced by decreased thymidine incorporation. Apoptotic changes in cells following drug treatment was confirmed by flow cytometry for Annexin and PI. Cleavage of caspases 3, 8 and 9 were confirmed on flow cytometry. Addition of the pan-caspase inhibitor zvad-fmk did not prevent drug-induced apoptosis confirming non-caspase mediated mechanisms of cell death as well. Primary myeloma cells from several patients were treated with increasing doses of the drug and IC50 similar to cell lines were seen in 8/10 patient samples tested. Interestingly, evaluation of U266 cell lines, which have a mix of CD45+ and negative cells as well as primary patient cells demonstrated more profound cytotoxicity and anti-proliferative activity of the drug on the CD45+ population relative to the CD45- cells. Immunoblotting studies demonstrated significant down regulation of IL-6 induced pSTAT3 with minor effects on the pERK and pAkt. The effect on pSAT3 was sustained compared to that on pERK and pAkt. This was accompanied by significant down regulation of Bcl-xL. Studies in a mouse model of myeloma are planned. Conclusion: These studies demonstrate significant in-vitro activity of JAK2 inhibition in multiple myeloma. In particular, the preferential targeting of CD45 cells, considered to reflect the proliferative compartment in myeloma holds out the promise for more sustained impact on the disease from a therapeutic standpoint. This is likely explained by the increased sensitivity of the CD45 cells to cytokines as a result of higher expression of different cytokine receptors as has been previously shown. This leads to increased activity of and dependence of the cells on the JAK-STAT pathway and likely explains the increased effect of the pathway inhibition. These studies form the framework for clinical evaluation of the drug in the setting of myeloma. Disclosures: Kumar: CELGENE: Research Funding; MILLENNIUM: Research Funding; BAYER: Research Funding; GENZYME: Research Funding; NOVARTIS: Research Funding.

Download Full-text

Validation of NMT1 and NMT2 As Novel Drug Targets in Adult Acute Myeloid Leukemia: Rationale for N-Myristoyltransferase Inhibition with Pclx-001 for Clinical Trials

Blood ◽

10.1182/blood-2021-154000 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4344-4344

Author(s):

Jay Gamma ◽

Aishwarya Iyer ◽

Megan Yap ◽

Zoulika Zak ◽

Krista Vincent ◽

...

Keyword(s):

Clinical Trials ◽

Acute Myeloid Leukemia ◽

Board Of Directors ◽

Cell Lines ◽

Peripheral Blood ◽

Myeloid Leukemia ◽

Research Funding ◽

Advisory Committees ◽

Acute Myeloid ◽

Privately Held

Abstract Background: N-terminal myristoylation is the transfer of the saturated fourteen carbon fatty acid myristate to an N-terminal glycine residue. This co- or post-translational protein modification promotes protein-protein and protein-lipid interactions and is essential for proper membrane localization and/or activity of up to 600 human intracellular proteins. N-myristoyltransferases (NMTs) are the enzymes responsible and two isoforms are found in humans. NMT1 (ubiquitous and essential for cell survival) and NMT2 (more variably expressed) differ in activity level and substrate specificities. NMT expression levels vary in some cancers, and with myristoylation being essential for activity of certain oncogenes including Src Family Kinases (SFKs). NMTs have therefore been proposed as an anti-cancer target. Dysregulation and oncogenic activity of SFKs occurs frequently in acute myeloid leukemia (AML), suggesting NMT inhibition could provide therapeutic benefit. PCLX-001 is a low nanomolar small molecule pan-NMT inhibitor with high oral bioavailability in clinical trials as once daily oral therapy for lymphoma and solid tumors. Methods and Results: Data from the TCGA Transcriptome database showed high NMT1 and low NMT2 were associated with reduced overall and event-free survival in adult AML, and high NMT1 - but not NMT2 - expression is associated with proliferative gene sets in AML cell lines. AML cell lines treated with PCLX-001 showed a significant reduction in total protein myristoylation, as well as reduced levels of SFK proteins and SFK phosphorylation. PCLX-001 induced apoptosis in AML cell lines and patient blasts at concentrations which spared a large proportion of peripheral blood lymphocytes and monocytes from healthy individuals. AML cell lines showed significant increase in BIP protein and ER stress in response to PCLX-001, along with caspase 3 cleavage. In an AML cell line derived xenograft (CDX) and two AML patient derived xenograft (PDX) series (n=1 DX for MV-4-11 and n=2 PDX), PCLX-001 monotherapy had dose-dependent anticancer activity and resulted in complete remissions in subcutaneous AML cell deposits. In tail-vein injection PDX models, PCLX-001 treatment resulted in up to 95% reduction of human CD45+ cells in peripheral blood and bone marrow. Conclusions: These findings validate NMT inhibition as a novel therapeutic strategy for AML. PCLX-001 preferentially targeted AML cells that rely on oncogenic activity of myristoylated proteins, inducing apoptosis and reducing leukemic burden. PCLX-001 warrants evaluation in clinical trials for adult AML. Disclosures Gamma: Pacylex Pharmaceuticals: Current holder of individual stocks in a privately-held company. Yap: Pacylex Pharmaceuticals: Current holder of individual stocks in a privately-held company, Patents & Royalties. Beauchamp: Pacylex Pharmaceuticals: Current Employment, Current holder of individual stocks in a privately-held company, Patents & Royalties. Mackey: Pacylex Pharmaceuticals, Inc.: Current holder of individual stocks in a privately-held company. Pemmaraju: Dan's House of Hope: Membership on an entity's Board of Directors or advisory committees; Abbvie Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; Aptitude Health: Consultancy; Sager Strong Foundation: Other; Celgene Corporation: Consultancy; LFB Biotechnologies: Consultancy; Plexxicon: Other, Research Funding; MustangBio: Consultancy, Other; Roche Diagnostics: Consultancy; Daiichi Sankyo, Inc.: Other, Research Funding; DAVA Oncology: Consultancy; Cellectis S.A. ADR: Other, Research Funding; Springer Science + Business Media: Other; Stemline Therapeutics, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; HemOnc Times/Oncology Times: Membership on an entity's Board of Directors or advisory committees; ASCO Leukemia Advisory Panel: Membership on an entity's Board of Directors or advisory committees; Samus: Other, Research Funding; ASH Communications Committee: Membership on an entity's Board of Directors or advisory committees; CareDx, Inc.: Consultancy; Novartis Pharmaceuticals: Consultancy, Other: Research Support, Research Funding; Incyte: Consultancy; Affymetrix: Consultancy, Research Funding; Protagonist Therapeutics, Inc.: Consultancy; Clearview Healthcare Partners: Consultancy; Blueprint Medicines: Consultancy; Bristol-Myers Squibb Co.: Consultancy; ImmunoGen, Inc: Consultancy; Pacylex Pharmaceuticals: Consultancy. Borthakur: Astex: Research Funding; Ryvu: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Protagonist: Consultancy; ArgenX: Membership on an entity's Board of Directors or advisory committees; University of Texas MD Anderson Cancer Center: Current Employment; Takeda: Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy. Brandwein: AbbVie: Honoraria; Jazz: Honoraria; Taiho: Honoraria; Astellas: Honoraria; Bristol Myers Squibb: Honoraria; Roche: Honoraria; Pfizer: Honoraria; Amgen: Honoraria. Berthiaume: Pacylex Pharmaceuticals, Inc.: Current holder of individual stocks in a privately-held company.

Download Full-text

AZD4320 Is a Novel and Potent BCL-2/XL Dual Inhibitor in Targeting Aggressive Mantle Cell Lymphoma

Blood ◽

10.1182/blood-2020-140775 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 44-44

Author(s):

Yijing Li ◽

Yang Liu ◽

Joseph McIntosh ◽

Alexa A Jordan ◽

Angela Leeming ◽

...

Keyword(s):

Cell Lines ◽

Cell Apoptosis ◽

Research Funding ◽

Cell Lymphoma ◽

Publicly Traded ◽

Dual Inhibitor ◽

Car T ◽

Mantle Cell

Introduction: Mantle cell lymphoma (MCL) is a rare subtype of B-cell non-Hodgkin's lymphoma. It is an incurable disease with frequent relapse from chemotherapies, targeted therapies, and cell therapies. Dysregulated expression of BCL-2 family members resulting in enhanced cell survival frequently occurs in many cancer types and often contributes to the development of therapeutic resistance. The BCL-2 inhibitor venetoclax has been shown to be effective in treating refractory/relapsed MCL patients. However, resistance often occurs and one of the underlying mechanisms of this resistance is the increased expression of other anti-apoptotic BCL-2 family members, such as BCL-XL and MCL-1. In this study, we assessed the in vitro and in vivo efficacy of a novel and highly potent BCL-2/XL dual inhibitor AZD4320 in preclinical models. Methods: Cell viability assay was tested after 72-hour treatment with AZD4320 in a panel of ibrutinib/venetoclax-sensitive and -resistant MCL cell lines by CellTiter-Glo (Promega). The assay was also done after a 24-hour treatment in primary PDX cells. Cell apoptosis assay was performed to determine if AZD4320 induces cell apoptosis in MCL cell lines. Furthermore, the in vivo efficacy of AZD4320 was assessed in a CAR-T resistant MCL patient-derived xenograft (PDX) model. Results: AZD4320 significantly inhibited cell proliferation in all tested MCL cell lines, including both ibrutinib/venetoclax-sensitive and -resistant cell lines. It had an IC50 value at a low nanomolar range between 0.59 nM to 18 nM. Consistently, AZD4320 was effective in targeting primary PDX cells ex vivo. AZD4320 induced cell apoptosis in a dose-dependent manner. AZD0466, the nanomedicine formulation of AZD4320 (30mg/kg, weekly, IV), dramatically inhibited tumor growth and prolonged mouse survival in an ibrutinib-CAR-T dual-resistant PDX mouse model. All mice tolerated the treatment dose without any body weight loss. Conclusion: The novel BCL-2/XL dual inhibitor AZD4320 demonstrated excellent anti-MCL activity in both ibrutinib/venetoclax-sensitive and -resistant MCL cells in vitro. This was further validated in vivo in a ibrutinib-CAR-T dual-resistant PDX model. These findings provide evidence that dual targeting of BCL-2 and BCL-XL by AZD4320 is promising as it may overcome therapeutic resistance in relapsed/refractory MCL. Disclosures Andersen: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Cidado:AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Wang:OMI: Honoraria, Other: Travel, accommodation, expenses; Nobel Insights: Consultancy; Loxo Oncology: Consultancy, Research Funding; Celgene: Consultancy, Other: Travel, accommodation, expenses, Research Funding; Kite Pharma: Consultancy, Other: Travel, accommodation, expenses, Research Funding; OncLive: Honoraria; Lu Daopei Medical Group: Honoraria; Acerta Pharma: Research Funding; VelosBio: Research Funding; BioInvent: Research Funding; Juno: Consultancy, Research Funding; Dava Oncology: Honoraria; Verastem: Research Funding; Molecular Templates: Research Funding; Oncternal: Consultancy, Research Funding; Pulse Biosciences: Consultancy; AstraZeneca: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Beijing Medical Award Foundation: Honoraria; Pharmacyclics: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; MoreHealth: Consultancy; Guidepoint Global: Consultancy; Targeted Oncology: Honoraria; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; InnoCare: Consultancy.

Download Full-text

Selective STAT3 Degraders Dissect Peripheral T-Cell Lymphomas Vulnerabilities Empowering Personalized Regimens

Blood ◽

10.1182/blood-2021-153995 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 865-865

Author(s):

Giuseppina Astone ◽

Luca Vincenzo Cappelli ◽

William Chiu ◽

Clarisse Kayembe ◽

Rui Wang ◽

...

Keyword(s):

Cell Cycle ◽

Flow Cytometry ◽

T Cell ◽

Growth Inhibition ◽

Research Funding ◽

Granzyme B ◽

Publicly Traded ◽

T Cell Lymphomas ◽

Peripheral T Cell Lymphomas

Abstract Introduction: Peripheral T-cell lymphomas (PTCLs) include heterogeneous entities of rare and aggressive neoplasms. The improved understanding of the biological/molecular mechanisms driving T-cell transformation and tumor maintenance has powerfully propelled new therapeutic programs. However, despite this progress, PTCLs remain an unmet medical need. Recurrent aberrations and the deregulated activation of distinct signaling pathways have been mapped and linked to selective subtypes. The JAK/STAT signaling pathway's deregulated activation plays a pathogenetic role in PTCL, including ALCL subtypes. STATs regulate the differentiation/phenotype, survival and cell-growth, metabolism, and drug resistance of T-cell lymphomas as well as host immunosuppressive microenvironments. Although many drugs' discovery programs were launched, a plethora of compounds has failed. Methods: We have discovered heterobifunctional molecules by an iterative medicinal chemistry SAR campaign that potently and selectively degrade STAT3 in a proteasome-dependent manner. Conventional PTCL cell lines and Patient Derived Tumor Xenograft (PDTX) and/or derived cell lines (PDTX-CL), carrying either WT- or mutated-STAT3, were exposed to increasing amounts (50nM⇒5µM) of STAT3-degraders. Proteins and mRNA transcripts (2⇒144hrs) were assessed by deep-proteomics and paired-end RNA sequencing, combined with WB/flow cytometry and qRT-PCR. Cell-titer-glo, cell titer blue, Annexin-V and S-cell cycle analyses were used as readouts. Chromatin accessibility, STAT3 DNA binding, 3D chromosomal architecture reorganization and 5-hmC profiling were assessed by ATACseq, CHIPseq and Hi-C and H3K27ac Hi-CHIP and mass-spectrometry. Drug testing/discovery combinations (96-well-plate) were performed using a semi-automated flow-cytometry. A battery of PTCL PDTX models were tested in pre-clinical trials. Results: Treatment of ALK+ ALCL (SU-DHL1) led to the rapid (~6hrs.) and profound down-regulation of STAT3 followed by the loss of canonical STAT3-regulated proteins (SOCS3, MYC, Granzyme B, GAS1, and IL2RA), without appreciable changes for other STAT family members (STAT1, STAT5b). In vitro, cytoplasmic, nuclear, and mitochondrial STAT3 downregulation was maintained up to 144 hrs. Loss of STAT3 in ALK+/- ALCL and BIA-ALCL cells was associated with major transcriptional changes (7116-10615 and 15114 DEGs in ALK- and ALK+ ALCL, respectively), underscoring public/shared as well as private time-dependent signatures. Main down-regulated pathways included JAK-STAT, MAPK, NF-kB, PI3K, TGFb, and TNFa. Comparison of STAT3 shRNA (ALK+ ALCL) and STAT3 degrader (ALK-/ALK+ ALCL) signatures demonstrated a substantial and concordant gene modulation (24hrs) among all models with the highest overlaps between ALK+ ALCL (Figure 3). To identify direct STAT3 gene targets, we analyzed CHIPseq peaks and predicted bindings sites, demonstrating that canonical genes, i.e., SOCS3, Granzyme B, GAS1, IL2RA, STAT3, and CD30, were significantly downregulated. Conversely, CD58, CD274, and MCH-I/II were upregulated at late time points. By mapping the STAT3 binding sites in ALK+ and ALK- ALCL, we have identified 1077 and 2763 STAT3 peaks within promoter/5'-/3'- and distant intergenic regions, corresponding to both coding and non-coding genes. Therapeutically, in vitro treatments led to cell cycle arrest and profound growth inhibition, and over time increased cell death of PTCL cells, including ALCL. Accordingly, growth inhibition of ALCL xenograft and PDTX tumors was also achieved (Figure 2). To identify drugs that could synergize withSTAT3-degrader activity, we tested a compound library (40) targeting pro-tumorigenic PTCL pathways as well as FDA-approved compounds. Ongoing studies are in progress. Conclusion: We have discovered selective STAT3 degraders which control PTCL growth. STAT3 degraders are powerful tools to define the STAT3 pathogenetic mechanisms and dissect genes/pathways to be targeted for T-cell lymphoma eradication. These data provide additional rationale for testing STAT3 degraders in the clinic for the treatment of aggressive malignancies including PTCL/ALCL. Figure 1 Figure 1. Disclosures Yang: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Sharma: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Dey: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Karnik: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Elemento: Owkin: Consultancy, Other: Current equity holder; Volastra Therapeutics: Consultancy, Other: Current equity holder, Research Funding; Johnson and Johnson: Research Funding; Eli Lilly: Research Funding; Janssen: Research Funding; Champions Oncology: Consultancy; Freenome: Consultancy, Other: Current equity holder in a privately-held company; One Three Biotech: Consultancy, Other: Current equity holder; AstraZeneca: Research Funding. Horwitz: Affimed: Research Funding; Aileron: Research Funding; ADC Therapeutics, Affimed, Aileron, Celgene, Daiichi Sankyo, Forty Seven, Inc., Kyowa Hakko Kirin, Millennium /Takeda, Seattle Genetics, Trillium Therapeutics, and Verastem/SecuraBio.: Consultancy, Research Funding; Acrotech Biopharma, Affimed, ADC Therapeutics, Astex, Merck, Portola Pharma, C4 Therapeutics, Celgene, Janssen, Kura Oncology, Kyowa Hakko Kirin, Myeloid Therapeutics, ONO Pharmaceuticals, Seattle Genetics, Shoreline Biosciences, Inc, Takeda, Trillium Th: Consultancy; Celgene: Research Funding; C4 Therapeutics: Consultancy; Crispr Therapeutics: Research Funding; Daiichi Sankyo: Research Funding; Forty Seven, Inc.: Research Funding; Kura Oncology: Consultancy; Kyowa Hakko Kirin: Consultancy, Research Funding; Millennium/Takeda: Research Funding; Myeloid Therapeutics: Consultancy; ONO Pharmaceuticals: Consultancy; Seattle Genetics: Consultancy, Research Funding; Secura Bio: Consultancy; Shoreline Biosciences, Inc.: Consultancy; Takeda: Consultancy; Trillium Therapeutics: Consultancy, Research Funding; Tubulis: Consultancy; Verastem/Securabio: Research Funding. DeSavi: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Liu: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company.

Download Full-text

Significant In Vitro Activity of a Novel Inhibitor of Apoptosis (IAP) Inhibitor LC161 In Multiple Myeloma

Blood ◽

10.1182/blood.v116.21.2998.2998 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2998-2998

Author(s):

Jessica Haug ◽

Vijay G. Ramakrishnan ◽

Teresa Kimlinger ◽

Timothy Halling ◽

Linda Wellik ◽

...

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

Signaling Pathways ◽

Cell Lines ◽

Research Funding ◽

In Vitro Activity ◽

Disease Biology ◽

Marker Proteins ◽

Dose Dependent

Abstract Abstract 2998 Background: Multiple myeloma remains incurable with current therapies and novel approaches based on disease biology are needed. Inhibitors of apoptosis (IAP) proteins represent a conserved group of proteins that are important regulators of apoptosis. X-linked IAP (XIAP) is the best studied IAP and inhibits pro-apoptotic caspases 3, 7 and 9. Multiple myeloma (MM) cell lines express high levels of XIAP and levels are further increased when stimulated by cytokines IL6 and IGF-1, both secreted in copious amounts in the myeloma microenvironment. IL6 and IGF1 up-regulate XIAP by activating the NF-κB, MAPK and PI3K signaling pathways that are commonly aberrant in MM and other tumors. XIAP mRNA contains an internal ribosomal entry site (IRES) sequence in the 5` untranslated region. IRES sequences enable direct ribosome recruitment and aid in translation that is independent of cap-mediated translation. Thus, molecules like mTOR inhibitors might not lead to XIAP downregulation. This was observed when rapamycin, an mTOR inhibitor, was used on MM cell lines. XIAP protein levels were not reduced due to the IRES sequence which leads to translation that is independent of the 5` cap and 4EBP1. Thus XIAP inhibitors might be able to overcome resistance associated with rapamycin treatment. Methods: LC161 was synthesized by Novartis Inc. (Basel, Switzerland). Stock solutions were made in DMSO, and subsequently diluted in RPMI-1640 medium for use. MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum (20% serum for primary patient cells) supplemented with L-Glutamine, penicillin, and streptomycin. Cytotoxicity was measured using the MTT viability assay and proliferation using thymidine uptake. Apoptosis was measured using flow cytometry upon cell staining with Annexin V-FITC and propidium iodide (PI). Immunoblotting was done on cell extracts at various time points following incubation with the drug in order to study the cell signaling pathways. Results: LC161 treatment resulted in a dose and time dependent inhibition of cell growth in the MM cell lines tested. Most of the cytotoxicity was evident by 72 hours, with minimal increase seen up to 96 hours of incubation. At 72 hours of incubation, the median inhibitory concentration varied considerably between various cell lines with an IC50 range of 2.5–25μ M. The IC50s were maintained when the cells were treated in co-culture with stromal cells or in the presence of IL6, IGF or VEGF. Dose-dependent decrease in proliferation of the cell lines was evidenced by decreased thymidine incorporation. Apoptotic changes in cells following drug treatment was confirmed by flow cytometry for Annexin and PI. Cleavage of caspases 3, 8 and 9 were confirmed on flow cytometry. Primary myeloma cells from patients were treated with increasing doses of the drug and dose dependent increase in apoptosis was observed. Immunoblotting studies demonstrated dose dependent significant down regulation of Xiap, cIAP1, cIAP2 and surviving and up-regulation of activated caspases 3, 8 and 9 and PARP. Furthermore, LC161 resulted in down regulation of pAkt, canonical and non-canonical NF-κB, pJNK, p-p38MAPK, c-Myc, Bcl-xL and Mcl1 and up-regulation of pErk and Bcl-2. We are currently examining basal levels of expression of the IAP proteins (Xiap, cIAP1 and cIAP2), pAkt and pErk in various MM cell lines to identify marker proteins that might predict response to this class of drug. In addition, our initial studies of LC161 in combination with the proteasome inhibitor bortezomib demonstrated synergy in killing MM cells in vitro. Additional combinations including inhibitors of the Akt/mTOR pathway and MEK/Erk pathway are currently been done. Conclusion: These studies demonstrate significant in vitro activity of LC161 in MM. Our results suggest the presence of two populations one very sensitive to IAP inhibition and one relatively less sensitive. Our current studies will help identify marker proteins that might predict response to LC161 treatment. We are currently testing LC261 in combination with known inhibitors of the other important signaling pathways implicated in MM disease biology as well as in-vivo experiments in mouse models. Performing these experiments will further validate the efficacy of LC161 as an anti-MM agent and form the basis for it to be taken up for clinical evaluation either as a single agent or in combination with other agent(s). Disclosures: Kumar: Celgene: Consultancy, Research Funding; Millennium: Research Funding; Merck: Consultancy, Research Funding; Novartis: Research Funding; Genzyme: Consultancy, Research Funding; Cephalon: Research Funding.

Download Full-text

Targeting MYD88-Mutant DLBCL with IRAKIMiDs: A Comparison to IRAK4 Kinase Inhibition and Evaluation of Synergy with Rational Combinations

Blood ◽

10.1182/blood-2020-135898 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 12-12

Author(s):

Jennifer Kimberly Lue ◽

John S Manavalan ◽

Christine Klaus ◽

Rahul Kanik ◽

Andre M. Grilo ◽

...

Keyword(s):

Flow Cytometry ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Kinase Inhibitors ◽

Dual Function ◽

Rna Seq ◽

Publicly Traded ◽

Advisory Committees ◽

Ic50 Values

Introduction: MYD88 mutations are found in 25% of DLBCL and are associated with an inferior survival. MYD88 is an adapter molecule, forming the core of the Myddosome complex. MYD88 mutations constitutively activate pathways such as NFқB, leading to lymphomagenesis. Essential to the Myddosome-dependent signaling pathway is the recruitment of IRAK4 which complexes with MYD88 to activate downstream effects. Targeting IRAK4 is therefore a rational therapeutic approach in MYD88-mutant lymphomas. First-in-class IRAKIMiDs, novel heterobifunctional degraders that target IRAK4 as well as the IMiD substrates Ikaros and Aiolos to enable the inhibition of both the NFkB and IRF4 pathways activated by MYD88 mutations, demonstrate potent efficacy in MYD88-mutant lymphomas (KTX-475, KTX-582, Walker D et al. AACR 2020). Herein, we compare the activity of IRAKIMiDs to IRAK4 kinase inhibitors and IMiDs alone in MYD88-mutant DLBCL, and evaluate rational combinations of IRAKIMiDs and other active agents in DLBCL for synergy. Methods: MYD88-mutant (n=4) and wild type (n=4) DLBCL cell lines were exposed to a panel of single agents (KTX-475, KTX-582, BAY1830839, CA-4948, CC-220, lenalidomide, pomalidomide, ibrutinib, umbralisib, venetoclax) in order to establish the drug concentration:cytotoxicity effect relationship. Cell viability was assessed using Celltiter-Glo assay at 24-hour intervals. IC50 values were computed. MYD88-mutant DLBCL cells were co-exposed to combinations of KTX-475 with venetoclax, ibrutinib, or umbralisib at concentrations representing their respective IC10-40 in order to determine synergy using the excess over bliss (EOB) method. Venetoclax, ibrutinib, and umbralisib were selected for combinational studies in order to target adverse pathways known to be associated in DLBCL biology. To confirm IRAK4 degradation, western blot and flow cytometry was performed. Apoptosis was evaluated with flow cytometry. Pre-treatment RNA-seq libraries were developed for the purpose of identifying GSEA and mutational analysis to predict response to IRAKIMiDs. Results: Exposure to IRAKIMiDs led to potent activity in MYD88-mutated DLBCL with IC50s in the low nanomolar range. IRAK4 degradation occured in a dose- and time-dependent manner and was observed as early as four hours after exposure. IRAKIMiDs induced superior cytotoxicity compared to two IRAK4 kinase inhibitors, including CA-4948 (Curis), which is currently under clinical investigation for relapsed/refractory NHL, as determined by lower IC50s in all cell lines. IRAKIMiD IC50s were also lower compared to pomalidomide, lenalidomide, and CC-220. KTX-475 was selected for synergy assessments based on IC50 values. Synergy was observed after exposure to KTX-475 in conjunction with venetoclax, ibrutinib, or umbralisib as determined by EOB >0 in the MYD88-mutant OCI-LY10 model, with maximum values peaking at 72-96 hours. After dual drug exposure, IRAK4 degradation was validated by flow cytometry demonstrating that the addition of venetoclax, ibrutinib or umbralisib to KTX-475 did not impair IRAK4 degradation capabilities. RNA-seq interpretation is currently underway. Conclusions: Collectively, our results demonstrate that dual-function degraders targeting both IRAK4 and the IMiD substrates Ikaros and Aiolos can serve as a potential therapeutic option for poor prognosis MYD88-mutant DLBCL. Our data thus far demonstrate improved efficacy of IRAKIMiDs compared to IRAK4 kinase inhibitors or IMiDs alone in vitro, as well as synergy with other active agents in combination regimens. A promising lead IRAKIMiD candidate has been identified, with initiation of a first-in-human clinical trial in B-cell lymphomas planned for 2021. Disclosures Lue: Daiichi Sankyo: Honoraria; AstraZeneca: Speakers Bureau; Astex Pharmaceuticals: Honoraria; Kymera Therapeutics: Honoraria, Research Funding; Kura Oncology: Honoraria. Klaus:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Kanik:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. McDonald:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Gollob:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Walker:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. O'Connor:Kymera Therapeutics: Current equity holder in private company, Honoraria, Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Current Employment, Current equity holder in publicly-traded company; Servier: Consultancy; Mundipharma: Other: Consulting; Astex Pharmaceuticals: Honoraria, Research Funding; Merck: Research Funding; Nomocan: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Other: Data Safety Monitoring Committee, Research Funding. Mainolfi:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company.

Download Full-text

Dual Targeting Bcl-2 By Venetoclax and Mcl-1 By AZD5991 to Overcome Therapeutic Resistance in Aggressive R/R Mantle Cell Lymphoma

Blood ◽

10.1182/blood-2021-151062 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1181-1181

Author(s):

Yijing Li ◽

Yang Liu ◽

Yuxuan Che ◽

Alexa A Jordan ◽

Joseph McIntosh ◽

...

Keyword(s):

Cell Lines ◽

Cell Apoptosis ◽

Research Funding ◽

Single Agent ◽

Therapeutic Resistance ◽

Publicly Traded ◽

Dual Targeting ◽

Car T ◽

Mantle Cell

Abstract Introduction Mantle cell lymphoma (MCL) is an aggressive subtype of non-Hodgkin's lymphoma. Frequent relapse from prior therapies remains a major medical challenge. BTK inhibitors (BTKi), such as ibrutinib and acalabrutinib, have demonstrated clinical benefit in MCL patients, however, resistance to BTKi is acquired by most MCL patients following initial response in the clinic. Unbalanced pro- and anti-apoptotic proteins have been shown to contribute to therapeutic resistance. Bcl-2 inhibitor venetoclax was approved by FDA to treat chronic lymphocytic leukemia, small lymphocytic lymphoma, or acute myeloid leukemia, and is currently under investigation in MCL patients. Venetoclax is efficacious with ORR up to 75% in treating BTKi-naive MCL patients and 53% BTKi-R/R patients. Interestingly, increased expression of Mcl-1 and Bcl-x L highly correlates with venetoclax resistance in MCL. AZD5991 is a highly selective Mcl-1 inhibitor and our preliminary data showed it is potent in targeting MCL cells (IC 50 =76-600 nM). Therefore, we hypothesize that dual targeting Bcl-2 by venetoclax and Mcl-1 by AZD5991 will achieve synergistic effect and significantly improve treatment outcome for aggressive R/R MCL patients. In this study, we assessed the synergistic efficacy of AZD5991 in combination with venetoclax in MCL preclinical models with R/R phenotype to ibrutinib, venetoclax or CD19 CAR T therapies. Methods Cell viability assay was performed to assess the in vitro efficacy of AZD5991 and venetoclax alone or in combination in a panel of ibrutinib/venetoclax-sensitive and -resistant MCL cell lines. Cell apoptosis assay was also performed to determine if AZD5991 and venetoclax induce cell death by cell apoptosis in MCL cell lines. Protein expression profiles of a panel of pro- and anti-apoptotic proteins and other relevant proteins were detected by western blots. In vivo efficacy of AZD5991 (30/30 mg/kg, intravenously, weekly) and venetoclax (5 mg/kg, oral, daily) alone or in combination was evaluated using PDX models derived from an ibrutinib-resistant patient or an ibrutinib-CAR-T dual-resistant MCL patient. Results AZD5991 and venetoclax combination potently and synergistically inhibited cell viability (combination index = 0.16-0.88) and enhanced cell apoptosis in vitro in both ibrutinib/venetoclax sensitive and resistant cell lines. Consistently, pro-apoptotic markers cleaved caspase 3 and cleaved PARP were increased. In an ibrutinib-resistant PDX mouse model, the combination of AZD5991 and venetoclax resulted in anti-MCL synergistic response. Consistently, the amount of β2M in the mouse plasma from the combo group was much lower than vehicle group (n =5, p = 0.010) and the two single agent groups (n =5, p = 0.005 and 0.013, respectively). In an ibrutinib-CAR-T dual-resistant PDX mouse model, co-treatment of AZD5991 and venetoclax inhibited tumor growth significantly and prolonged mouse survival for at least 100 days compared to vehicle or either single agent. All mice tolerated the treatment dose without any weight loss. Conclusion Significant anti-MCL synergy was observed for AZD5991 and venetoclax combo in vitro in both pairs of Jeko-1/JeKo-ibrutinib-R and Mino/Mino-venetoclax-R. This was further validated in aggressive R/R PDX models including an ibrutinib-resistant PDX model and an ibrutinib-CD19 CAR-T dual-resistant PDX model. Altogether, these findings suggest that dual targeting Bcl-2 and Mcl-1 is synergistic and has the potential in overcoming multiple acquired resistance phenotypes, including CD19 CAR T-cell therapy. These data provide insights on therapeutic development to improve patient outcome by overcoming therapeutic resistance. Disclosures Andersen: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Cidado: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Wang: Clinical Care Options: Honoraria; VelosBio: Consultancy, Research Funding; BioInvent: Research Funding; Genentech: Consultancy; Celgene: Research Funding; Molecular Templates: Research Funding; Dava Oncology: Honoraria; BGICS: Honoraria; Imedex: Honoraria; BeiGene: Consultancy, Honoraria, Research Funding; Juno: Consultancy, Research Funding; Newbridge Pharmaceuticals: Honoraria; CAHON: Honoraria; Kite Pharma: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Bayer Healthcare: Consultancy; DTRM Biopharma (Cayman) Limited: Consultancy; InnoCare: Consultancy, Research Funding; Moffit Cancer Center: Honoraria; Hebei Cancer Prevention Federation: Honoraria; Scripps: Honoraria; Mumbai Hematology Group: Honoraria; OMI: Honoraria; Epizyme: Consultancy, Honoraria; Chinese Medical Association: Honoraria; Oncternal: Consultancy, Research Funding; The First Afflicted Hospital of Zhejiang University: Honoraria; Anticancer Association: Honoraria; AstraZeneca: Consultancy, Honoraria, Research Funding; Miltenyi Biomedicine GmbH: Consultancy, Honoraria; Acerta Pharma: Consultancy, Honoraria, Research Funding; CStone: Consultancy; Loxo Oncology: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Physicians Education Resources (PER): Honoraria; Lilly: Research Funding.

Download Full-text

Resistance to Complement Dependent Cytotoxicity in CLL Cells From Patients Treated with Ofatumumab

Blood ◽

10.1182/blood.v118.21.2836.2836 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2836-2836

Author(s):

Nisar A. Baig ◽

Ronald P. Taylor ◽

Margaret A. Lindorfer ◽

Amy K Church ◽

Betsy R. LaPlant ◽

...

Keyword(s):

Flow Cytometry ◽

Research Funding ◽

Viable Cell ◽

Serum Samples ◽

Fluorescent Intensity ◽

Cell Counts ◽

Complement Dependent Cytotoxicity ◽

Low Levels

Abstract Abstract 2836 Monoclonal antibodies (mAb) are an effective but non-curative treatment of CLL. Although the mechanisms of action of mAb in vivo are not fully defined, complement dependent cytotoxicity (CDC) appears to play an important role. We have previously shown that addition of ofatumumab (OFA) significantly increases alemtuzumab (ALM) CDC in vitro and identified a subpopulation of CLL cells that are intrinsically resistant to activated complement. We propose that resistance to CDC could be an important cause of mAb treatment failure in CLL patients. To test the hypothesis that CLL cells treated in vivo with OFA would be resistant to subsequent OFA retreatment in vitro, we studied CLL cells and serum from 10 previously untreated patients with progressive CLL treated with pentostatin, cyclophosphamide, and ofatumumab (PCO). Methods. Patients received 300 mg of OFA followed by 2 mg/m2 of pentostatin and 600 mg/m2 of cyclophosphamide on day 1 of therapy. Samples were taken prior to treatment (S1), immediately after the OFA infusion and prior to chemotherapy (S2), and post-chemotherapy before the second dose of OFA on day 2 (S3). Mononuclear blood cells isolated from EDTA anticoagulated blood by Ficoll-Paque centrifugation were purified to achieve a concentration of > 90% CLL. CDC was assayed at a concentration of 2 × 106/ml CLL cells with 10 mg/ml of ALM (Genzyme), OFA (G.S.K.) or rituximab (RTX)(Genentech) in AIM V medium (Invitrogen, CA) and 10% normal human serum (10%NHS)(Sigma, MO) as a source of complement for 1 hour at 37°C. Absolute viable cell counts were measured by flow cytometry using counting beads (Trucount, BD, CA) and propidium iodide staining (Sigma, MO) with a FACSCalibur (BD, CA) and CellQuest Pro software (BD, CA). Percent CDC was calculated relative to counts for CLL cells treated with only 10%NHS. Binding of mAb and C3b to CLL cells was measured by flow cytometry with mouse anti-human Fc antibody FITC-HB43 and anti-C3b antibody FITC-7C12. C5 deficient serum (C5-serum)(Sigma, MO) was used to examine mAb and C3b binding without CDC. Results were expressed as delta mean fluorescent intensity (dMFI) relative to cells treated without mAb in 10%NHS or C5-serum as appropriate. Complement (CH50) was measured based on lysis of opsonized sheep red cells using standard methods. OFA concentrations in serum samples were determined by measuring the level of binding to Daudi cells compared to standards as previously described. Results: S1 CLL cells were significantly more susceptible to in vitro OFA CDC (median 30%) than S2 and S3 cells (both 0%, p=0.002)(Fig. 1). In contrast, high levels of CDC (median 86 – 88%) were induced in vitro by ALM in all these samples and prior in vivo exposure to OFA did not affect susceptibility to ALM CDC in vitro (p>0.1). CLL cells from S2&S3 had low levels of OFA binding both prior to and post in vitro exposure to OFA (median dMFI 2 and 3 respectively) which was significantly less than OFA binding for S1 (median dMFI 38 p=0.002) likely reflecting in vivo trogocytosis of bound OFA and CD20, as previously described for RTX. In vitro ALM binding was higher than OFA (median dMFI>150) in all specimens. C3b binding was low in S2&S3 both before and after in vitro OFA exposure (median dMFI 3 and 4 respectively) with significantly higher levels in S1 (median dMFI 94, p=0.002). C3b binding was higher in all CLL cells treated with ALM (median dMFI>358). OFA treatment resulted in marked decreases in serum complement levels in S2 (median 86%, range 65–98%) and S3 (median 78% range 61–88%) compared to S1. The median serum OFA concentration was 18.5 μg/ml (range 7.3–50.4) in S2 and 19.8 μg/ml (range 0–32.3) in S3. Conclusions: Circulating CLL cells from patients treated with OFA are resistant to in vitro OFA CDC primarily because of low levels of expression of CD20. The reduction in complement titers could limit in vivo CDC but we found no evidence that low levels of OFA were important. Cells surviving OFA in vivo do retain sensitivity to ALM CDC. Our data support the previous description of trogocytosis in CLL patients treated with RTX and suggest that lower doses of these mAb, that promote far lower levels of trogocytosis, could be more effective in sustaining CDC. In addition, our study provides pre-clinical data to support a clinical trial of combination therapy with OFA and ALM for CLL. Acknowledgment: This study was supported by funding from GlaxoSmithKline and the University of Iowa/Mayo Clinic Lymphoma SPORE (CA097274). Disclosures: Taylor: Genmab: Consultancy; Glaxo Smith Kline: Research Funding. Zent:GlaxoSmithKline: Research Funding; Genentech: Research Funding; Genzyme: Research Funding.

Download Full-text