Activity of New Heat Shock Protein 90 (hsp90) Inhibitor NVP-AUY922 Against Myeloma Cells Sensitive and Resistant to Conventional Agents.

Abstract Context: The molecular chaperone hsp90 is a major anti-cancer therapeutic target because it regulates the function of proteins with pivotal roles in tumor cell proliferation, survival and drug resistance, including mutated/chimeric oncoproteins or oncogenic kinases/receptors. Our preclinical studies on the ansamycin hsp90 inhibitor tanespimycin (17-AAG) provided the rationale for clinical trials, either alone or in combination with the proteasome inhibitor bortezomib, for treatment of relapsed/refractory MM. In this study, we report preclinical studies of the new, non-ansamycin, hsp90 inhibitor NVP-AUY922. Methods/Results: We tested 36 human MM cell lines and observed with MTT colorimetric survival assays potent time- and dose-dependent anti-MM activity of NVP-AUY922. IC50 values were <12.5 nM for 31 of 35 cell lines, which were also more sensitive than all non-malignant cells tested with NVP-AUY922, including bone marrow stromal cells (BMSCs), immunortalized human hepatocytes and normal donor PBMCs, indicating a differential selectivity of NVP-AUY922 against neoplastic tissues vs. non-malignant cells. Importantly, MM cell lines or primary MM tumor cells resistant to dexamethasone (Dex), melphalan, immunomodulatory thalidomide derivatives (IMIDs), bortezomib or TRAIL were sensitive to NVP-AUY922. In addition, MM-1S cells constitutively over-expressing Akt were equally sensitive to NVP-AUY922 compared to their parental cell line. These anti-MM effects were rapid, as MM cells were committed to cell death within 16 hrs of exposure to only 25 nM of NVP-AUY922. NVP-AUY922 overcame the protective effects conferred to MM cells by exogenous IL-6 and IGF-1, while BMSC co-culture attenuated NVP-AUY922 activity only at doses <20 nM, as evidenced by compartment-specific bioluminescence imaging (CS-BLI). Mechanistic studies of NVP-AUY922 treatment of MM-1S cells showed early G2/M arrest followed by increased cell death, accompanied by caspase-3 and -8 cleavage (as early as 16 hrs). These events were preceded by decreased levels of Akt, B-Raf, phospho-MEK, cIAP2, and XIAP; and compensatory upregulation of hsp27 and hsp70. Given the highly single-agent activity of NVP-AUY922 at even low nM concentrations, formal statistical documentation of synergy was not observed in NVP-AUY922 combinations with conventional (e.g. Dex, doxorubicin) or novel (e.g. bortezomib) anti-MM agents. Encouragingly, no evidence of antagonism with any of these combinations was observed, indicating that NVP-AUY922 can be combined with current anti-MM agents in clinical settings. Conclusion: The new hsp90 inhibitor NVP-AUY922 has potent in vitro activity against MM cells resistant to conventional therapeutics, with selectivity for malignant compared to normal cells. Ongoing in vivo experiments and studies to identify biomarkers of pronounced sensitivity to NVP-AUY922 will help provide a framework for potential clinical trials of NVP-AUY922 in MM and other neoplasias.

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Targeting BCL-2 in Cancer: Advances, Challenges, and Perspectives

Cancers ◽

10.3390/cancers13061292 ◽

2021 ◽

Vol 13 (6) ◽

pp. 1292 ◽

Cited By ~ 1

Author(s):

Shirin Hafezi ◽

Mohamed Rahmani

Keyword(s):

Drug Resistance ◽

Cell Death ◽

Targeted Therapies ◽

Single Agent ◽

Therapeutic Agents ◽

Malignant Cells ◽

Future Perspectives ◽

Comprehensive Overview ◽

Antiapoptotic Proteins ◽

Preclinical And Clinical Studies

The major form of cell death in normal as well as malignant cells is apoptosis, which is a programmed process highly regulated by the BCL-2 family of proteins. This includes the antiapoptotic proteins (BCL-2, BCL-XL, MCL-1, BCLW, and BFL-1) and the proapoptotic proteins, which can be divided into two groups: the effectors (BAX, BAK, and BOK) and the BH3-only proteins (BIM, BAD, NOXA, PUMA, BID, BIK, HRK). Notably, the BCL-2 antiapoptotic proteins are often overexpressed in malignant cells. While this offers survival advantages to malignant cells and strengthens their drug resistance capacity, it also offers opportunities for novel targeted therapies that selectively kill such cells. This review provides a comprehensive overview of the extensive preclinical and clinical studies targeting BCL-2 proteins with various BCL-2 proteins inhibitors with emphasis on venetoclax as a single agent, as well as in combination with other therapeutic agents. This review also discusses recent advances, challenges focusing on drug resistance, and future perspectives for effective targeting the Bcl-2 family of proteins in cancer.

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Combined Inhibition of AKT and KIT Restores Expression of Programmed Cell Death 4 (PDCD4) in Gastrointestinal Stromal Tumor

Cancers ◽

10.3390/cancers13153699 ◽

2021 ◽

Vol 13 (15) ◽

pp. 3699

Author(s):

Marya Kozinova ◽

Shalina Joshi ◽

Shuai Ye ◽

Martin G. Belinsky ◽

Dinara Sharipova ◽

...

Keyword(s):

Cell Death ◽

Gastrointestinal Stromal Tumor ◽

Cell Lines ◽

Synergistic Effects ◽

Single Agent ◽

Xenograft Model ◽

Stromal Tumor ◽

In Vivo Studies ◽

Preclinical Model ◽

Kit Mutation

The majority of gastrointestinal stromal tumor (GIST) patients develop resistance to the first-line KIT inhibitor, imatinib mesylate (IM), through acquisition of secondary mutations in KIT or bypass signaling pathway activation. In addition to KIT, AKT is a relevant target for inhibition, since the PI3K/AKT pathway is crucial for IM-resistant GIST survival. We evaluated the activity of a novel pan-AKT inhibitor, MK-4440 (formerly ARQ 751), as monotherapy and in combination with IM in GIST cell lines and preclinical models with varying IM sensitivities. Dual inhibition of KIT and AKT demonstrated synergistic effects in IM-sensitive and -resistant GIST cell lines. Proteomic analyses revealed upregulation of the tumor suppressor, PDCD4, in combination treated cells. Enhanced PDCD4 expression correlated to increased cell death. In vivo studies revealed superior efficacy of MK-4440/IM combination in an IM-sensitive preclinical model of GIST compared with either single agent. The combination demonstrated limited efficacy in two IM-resistant models, including a GIST patient-derived xenograft model possessing an exon 9 KIT mutation. These studies provide strong rationale for further use of AKT inhibition in combination with IM in primary GIST; however, alternative agents will need to be tested in combination with AKT inhibition in the resistant setting.

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Targeting Sphingolipids for Cancer Therapy

Frontiers in Oncology ◽

10.3389/fonc.2021.745092 ◽

2021 ◽

Vol 11 ◽

Author(s):

Osmel Companioni ◽

Cristina Mir ◽

Yoelsis Garcia-Mayea ◽

Matilde E. LLeonart

Keyword(s):

Clinical Trials ◽

Cell Death ◽

Cancer Therapy ◽

Preclinical Studies ◽

Hepatic Toxicity ◽

Multiple Cancers ◽

Extensive Class ◽

Effective Drugs

Sphingolipids are an extensive class of lipids with different functions in the cell, ranging from proliferation to cell death. Sphingolipids are modified in multiple cancers and are responsible for tumor proliferation, progression, and metastasis. Several inhibitors or activators of sphingolipid signaling, such as fenretinide, safingol, ABC294640, ceramide nanoliposomes (CNLs), SKI-II, α-galactosylceramide, fingolimod, and sonepcizumab, have been described. The objective of this review was to analyze the results from preclinical and clinical trials of these drugs for the treatment of cancer. Sphingolipid-targeting drugs have been tested alone or in combination with chemotherapy, exhibiting antitumor activity alone and in synergism with chemotherapy in vitro and in vivo. As a consequence of treatments, the most frequent mechanism of cell death is apoptosis, followed by autophagy. Aslthough all these drugs have produced good results in preclinical studies of multiple cancers, the outcomes of clinical trials have not been similar. The most effective drugs are fenretinide and α-galactosylceramide (α-GalCer). In contrast, minor adverse effects restricted to a few subjects and hepatic toxicity have been observed in clinical trials of ABC294640 and safingol, respectively. In the case of CNLs, SKI-II, fingolimod and sonepcizumab there are some limitations and absence of enough clinical studies to demonstrate a benefit. The effectiveness or lack of a major therapeutic effect of sphingolipid modulation by some drugs as a cancer therapy and other aspects related to their mechanism of action are discussed in this review.

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Pan-BCL-2 Family Small Molecule Inhibitor GX015-070 (GeminX, Inc.) Exhibits Single Agent Cytotoxicity, Is Synergistic with Select Anti-Leukemia Cytotoxic Agents, and Induces Apoptosis in Cell Lines with t(4;11)(q21;q23) Translocations.

Blood ◽

10.1182/blood.v106.11.3368.3368 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3368-3368 ◽

Cited By ~ 1

Author(s):

Jessicca M. Rege ◽

Blaine W. Robinson ◽

Manish Gupta ◽

Jeffrey S. Barrett ◽

Peter C. Adamson ◽

...

Keyword(s):

Cell Death ◽

Western Blot ◽

Family Member ◽

Cell Lines ◽

Blot Analysis ◽

Western Blot Analysis ◽

Caspase 3 ◽

Single Agent ◽

Cytotoxic Agents ◽

Response Surface Modeling

Abstract Background: Leukemias with MLL translocations, especially t(4;11), often are resistant to common chemotherapeutic agents, which may be due to abnormal apoptosis regulation. Pro- and anti-apoptotic BCL-2 family member interactions govern initiation of the intrinsic apoptosis pathway. GX015-070, which currently is in Phase I/IIA clinical trials, mimics the BH3 domain on pro-apoptotic BCL-2 family proteins and can bind the BH3 binding pocket of anti-apoptotic BCL-2 family members and modulate apoptosis. We performed comprehensive protein expression profiling of BCL-2 family member proteins and evaluated in vitro activity and mechanism of action of GX015-070 in cell lines with t(4;11). Methods: Baseline expression of BCL-2 family proteins was determined by Western blot analysis. Cytotoxicity was assessed by MTT after a 3 day exposure of RS4:11, SEM-K2 and MV4-11 cells in log phase growth to single agent GX015-070 at concentrations from 5 nM to 7.5 μM. Combined effects of fixed-concentration GX015-070 with cytotoxic agents over a range of concentrations were assayed by MTT, and the results were analyzed by pharmacostatistical response surface modeling. Disruption of specific pro- and anti-apoptotic BCL-2 family member interactions was investigated by co-immunoprecipitation/Western blot analysis. Flow cytometry and/or Western blot analysis of Caspase-3 activation, and a FACS TUNEL assay, were used to assess apoptosis in GX015-070 treated and untreated cells. Results: The three cell lines had similar baseline levels of expression of BCL-2 family proteins. BCL-2 and BAX were most abundant followed by PUMA, BAK, BCL-XL, BIM-EL, MCL-1, BIK and NOXA. Results of assays of GX015-070 activity and mechanism of action are in shown in the table. Conclusions: These data indicate that GX015-070 has potent cytotoxic activity in cell lines with t(4;11) as a single agent and that the cytotoxicity results from apoptosis. Response surface modeling in RS4:11 cells suggested ability to achieve effective doses with GX015-070 combined with cytosine arabinoside (Ara-C), dexamethasone (Dex) or doxorubicin (ADR) that are lower than projected from the single agents, but synergy was not suggested when GX015-070 was combined with etoposide, methotrexate or 6-thioguanine. The co-IP experiments give proof of principle that GX015-070 disrupts pro- and anti-apoptotic BCL-2 family protein interactions in cell lines with t(4;11). Additional pre-clinical experiments directed at overcoming drug resistance from abnormal cell death regulation in leukemias with t(4;11) using GX015-070 are in progress. These studies provide a framework to understand the cell death/survival machinery in primary leukemias with t(4;11) translocations more completely and manipulate that machinery to achieve better treatments. GX015-070 Activity and Mechanism Cell Line Single Agent Activity Synergy Inhibition Caspase-3 Activation TUNEL RS4:11 IC50=43.5 nM Ara-C, Dex, ADR Mcl1:Bak; Bcl2:Bak + + SEM-K2 IC50=156 nM In progress Mcl1:Bak; Bcl2:Bak + In Progress MV4-11 IC50=123 nM In progress Mcl1:Bak In progress +

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Transiently Elevated AST/LDH Are Associated with Clinical Response to Recombinant Circularly Permuted TRAIL (CPT) Plus Thalidomide in Patients with Relapsed and/or Refractory Multiple Myeloma

Blood ◽

10.1182/blood.v124.21.3478.3478 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3478-3478

Author(s):

Wenming Chen ◽

Peng Wei ◽

Shifang Yang ◽

Xiangjun Zheng ◽

Lugui Qiu ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Death ◽

Cell Lines ◽

Time Course ◽

Single Agent ◽

Release Rates ◽

Refractory Multiple Myeloma ◽

Before And After ◽

Clinical Responses ◽

Thalidomide Treatment

Abstract Introduction: Circularly permuted TRAIL (CPT), a recombinant mutant of human Apo2L/TRAIL, is a promising anti-tumor candidate. In previous phase1/2 clinical trials of single-agent CPT in patients with relapsed and/or refractory multiple myeloma (RRMM), transient elevations of serum AST and LDH were observed early after CPT treatment in most response patients, but not in the non-respondent. Positive correlations were found between increased AST/LDH on day 2 or 3 (24 or 48 hours) after CPT initial dosing and the clinical responses to CPT. Objective: To determine whether transiently elevated AST/LDH is predictive of responses to CPT plus thalidomide in thalidomide-relapsed or refractory MM patient and the time course of AST or LDH elevation. Methods: We retrospectively analyzed the data of a phase 2 study of CPT plus thalidomide. The changes of serum AST, LDH or ALT were analyzed before treatment and on days 2, 3 and 6 after initial dosing. Relationship was evaluated between ΔAST, ΔLDH, ΔALT (ratio of ASTD2, LDHD2 or ALTD2to baseline value) and the best clinical responses. Four MM cell lines (RPMI 8226, NCI-H929, MM.1S, MM.1R) sensitive to CPT were used to detect the concentrations of AST, ALT and LDH in the cytoplasm or the medium of CPT-treated cells, with the purpose of determining whether CPT-induced cell death could result in an elevation of AST or LDH. Results: Of 41 efficacy-evaluable patients, 9(22.0%) achieved a partial response (PR) or better and 14 (34.1%) achieved a minimal response (MR) or better. The serum ASTD2 and LDHD2 levels were dramatically increased from baseline in patients with ≥PR or ≥MR, but not in those with NC/PD. However, serum ALTD2 was comparable to baseline value either in response patients (≥PR or ≥MR) or in non-response ones (NC/PD). Consequently, the median ΔAST or ΔLDH of patients with ≥PR or ≥MR was significantly higher than that of patients with NC/PD (Table 1). The elevation of AST or LDH was transient with a peak on day 2 after treatment, then dramatically declined on day 3, and usually disappeared within one week (at most two weeks for LDH) (Figure 1), which was uniquely observed in the first treatment cycle. A univariate logistic-regression analysis showed that ΔASTwas predictive of achieving responses of ≥MR or not (P=0.04). Indeed, patients with higher level of ΔAST had higher probability of achieving responses of ≥MR (72.7% in patients with ΔAST>1.35 vs. 26.3% in patients with ΔAST≤1.35). In the cytoplasm of MM cell lines, abundant AST and LDH but only detectable level of ALT was observed. There was no significant change in the release rates of AST and LDH with CPT incubation for 1, 2, 3, 4 or 6 hours, even though the cell viabilities at 6h had already declined to about 10-20% of the control cells by ATP chemiluminescent assay. Remarkable increase in the release rates of AST and LDH occurred at 24h, with no evident change of ALT, which was consistent with what was observed in patients in the clinical study. It was suggested that the transient elevation of AST or LDH in RRMM patients was most likely resulted from CPT-induced myeloma cell death. Conclusion: The early transient elevations of serum AST and LDH after CPT plus thalidomide treatment were positively correlated with the clinical responses to CPT plus thalidomide, and were possibly resulted from CPT-induced cell death. ΔAST on day2 could be a surrogate response biomarker for CPT treatment in RRMM patients. Figure 1 Representative time course of AST, ALT or LDH changes before and after CPT plus thalidomide treatment in response patients Figure 1. Representative time course of AST, ALT or LDH changes before and after CPT plus thalidomide treatment in response patients Table 1 Differences in ΔAST, ΔALT and ΔLDH between response patients (≥PR or ≥MR) and non-response patients (NC/PD) Table 1. Differences in ΔAST, ΔALT and ΔLDH between response patients (≥PR or ≥MR) and non-response patients (NC/PD) Disclosures Wei: Beijing Sunbio Biotech Co., Ltd.: Employment. Yang:Beijing Sunbio Biotech Co., Ltd.: Employment. Zheng:Beijing Sunbio Biotech Co., Ltd.: Employment. Pang:Beijing Sunbio Biotech Co., Ltd.: Employment.

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The combination of the farnesyl transferase inhibitor lonafarnib and the proteasome inhibitor bortezomib induces synergistic apoptosis in human myeloma cells that is associated with down-regulation of p-AKT

Blood ◽

10.1182/blood-2005-06-2584 ◽

2005 ◽

Vol 106 (13) ◽

pp. 4322-4329 ◽

Cited By ~ 41

Author(s):

Ebenezer David ◽

Shi-Yong Sun ◽

Edmund K. Waller ◽

Jing Chen ◽

Fadlo R. Khuri ◽

...

Keyword(s):

Cell Death ◽

Combination Therapy ◽

Cell Lines ◽

Proteasome Inhibitor ◽

Single Agent ◽

Myeloma Cell ◽

Tumor Cell Death ◽

Down Regulation ◽

Farnesyl Transferase ◽

Farnesyl Transferase Inhibitor

The identification of signaling pathways critical to myeloma growth and progression has yielded an array of novel agents with clinical activity. Multiple myeloma (MM) growth is IL-6 dependent, and IL-6 is secreted in an autocrine/paracrine fashion with signaling via the Ras/Raf/mitogen-activated protein kinase (MAPK) pathway. We hypothesized that combining a Ras pathway inhibitor (lonafarnib, SCH66336) with a proteasome inhibitor (bortezomib, Velcade, PS-341) would enhance myeloma-cell killing. MM cell lines and primary human cells were used to test either single agent bortezomib, lonafarnib, or the combination on MM signaling and apoptosis. Combination therapy induced synergistic tumor-cell death in MM cell lines and primary MM plasma cells. Cell death was rapid and associated with increased caspase 3, 8, and 9 cleavage and concomitant down-regulation of p-AKT. Down-regulation of p-AKT was seen only in combination therapy and not seen with either single agent. Cells transfected with constitutively active p-AKT, wild-type AKT, or Bcl-2 continued to demonstrate synergistic cell death in response to the combination. The order of addition was critically important, supporting bortezomib followed by lonafarnib as the optimal schedule. The combination of a proteasome inhibitor and farnesyl transferase inhibitor demonstrates synergistic myeloma-cell death and warrants further preclinical and clinical studies.

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BCR-ABL+ Bp-ALL Exhibits Heightened Sensitivity to Simultaneous Induction of Energy and ER Stress Via Downregulation of Mcl-1 Expression

Blood ◽

10.1182/blood.v124.21.916.916 ◽

2014 ◽

Vol 124 (21) ◽

pp. 916-916

Author(s):

Guy J. Leclerc ◽

Joanna DeSalvo ◽

Gilles M. Leclerc ◽

Theodore J. Lampidis ◽

Julio C. Barredo

Keyword(s):

Cell Death ◽

Er Stress ◽

Cell Lines ◽

Aerobic Glycolysis ◽

Lymphoblastic Leukemia ◽

Single Agent ◽

Mtor Inhibition ◽

Energy Stress ◽

Simultaneous Induction ◽

And Control

Abstract Tumor metabolism has emerged as a hallmark of cancer by which the oncogenic profile of cancer cells pairs energy availability with growth and survival. BCR-ABL+ acute lymphoblastic leukemia (BCR-ABL+ ALL) accounts for 25% of ALL and the BCR-ABL fusion protein has been correlated with alterations in glucose metabolism. The glucose analogue 2-deoxy-D-glucose (2DG), induces simultaneous energy and ER-stress by inhibiting glycolysis (energy stress) via blocking HK and PGI enzymes, and interfering with N-linked glycosylation through its incorporation in place of mannose into the lipid-linked oligosaccharide (LLO) chain leading to premature termination of LLO synthesis (ER stress). We recently demonstrated that inhibition of both aerobic glycolysis and N-linked glycosylation by 2DG led to apoptosis in ALL (Mol Cancer Res 10:969, 2012). Among ALL subtypes, BCR-ABL+ ALL cell lines SupB15 and TOM1 exhibited the highest sensitivity to 2DG suggesting that in ALL cells BCR-ABL expression may be linked to heightened sensitivity to energy and/or ER stress. To test this premise, we constructed NALM6 (Bp-ALL) stable cell lines expressing the BCR-ABL p190 fusion and found that expression of the BCR-ABL p190 fusion in NALM6 cells significantly increased 2DG-induced apoptosis compared to mock transfected controls. To investigate the contribution of each form of stress (energy and ER stress) in the increased sensitivity of BCR-ABL+ cells to 2DG, we examined the effects of agents known to preferentially induce energy stress (lowering ATP) such as 2-fluoro-2-deoxy-D-glucose and oxamate, and ER stress such as tunicamycin. We found no differences in cell death or ATP levels between BCR-ABL+ and control ALL cells treated with agents solely inducing energy or ER stress, indicating the increased susceptibility of BCR-ABL+ ALL cells to 2DG results from the simultaneous induction of both types of stress. Consistent with these data, Fluorophore-Assisted Carbohydrate Electrophoresis and immunoblotting demonstrated that 2DG induced equivalent inhibition of N-linked glycosylation and upregulation of the UPR signaling in BCR-ABL+ and control ALL cells. Similar findings were seen in primary ALL patient samples. Consequently, simultaneous energy and ER stress are required for the heightened vulnerability to these forms of stress conferred by BCR-ABL in ALL. Based on the known function of Mcl-1 promoting survival in BCR-ABL+ ALL, we investigated its role in the mechanism of cell death induced by energy and/or ER stress agents. We uncovered that BCR-ABL+ ALL cell lines exhibited the lowest levels of Mcl-1 expression compared to control BCR-ABL- ALL and CML cells, and this basal level of Mcl-1 expression directly correlated with their resistance to energy and/or ER stressing agents. Further, we found that Mcl-1 knock-down by shRNA sensitized ALL and CML cells to the dual induction of energy and ER stress by 2DG. We also found that Mcl-1 expression was further downregulated in BCR-ABL+ and - ALL cells treated with energy and/or ER stressors which correlated with the level of apoptosis. Using qRT-PCR or co-treatment with the pancaspase inhibitor qVD-OPH or the proteasome inhibitor bortezomib, we showed that Mcl-1 downregulation by energy and/or ER stressors is neither due to transcriptional, caspase dependent cleavage, or post-translational regulation mechanisms but likely due to translational control of the Mcl-1 protein via AMPK/mTOR and the UPR PERK/p-eIF2α. Finally, because TKIs lead to decrease ATP (energy stress) in BCR-ABL+ cells, we co-treated BCR-ABL+ ALL cells with TKIs plus ER stressors and found that the simultaneous induction of these forms of stress by these combinations significantly increased cell death compared to single drug. Further, we found that TKIs + ER stressors further downregulated Mcl-1 and GRP78 expression compared to single agent treatment. Taken together, our data demonstrate that simultaneous induction of energy and ER stress leads to significant induction of apoptosis in BCR-ABL+ ALL cells, and these heightened vulnerability to these forms of stress is likely mediated by downregulation of Mcl-1 expression via AMPK activation/mTOR inhibition and PERK/p-eIF2α. Our study demonstrates that strategies with agents that induce energy and/or ER stress plus BCR-ABL TKIs warrant further consideration as a potential approach for future clinical translation into trials for BCR-ABL+ ALL patients. Disclosures No relevant conflicts of interest to declare.

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SAR650984 (SAR) Directly Promotes Homotypic Adhesion-Related Multiple Myeloma (MM) Cell Death and SAR-Induced Anti-MM Activities Are Enhanced By Pomalidomide, More Potently Than Lenalidomide

Blood ◽

10.1182/blood.v124.21.2124.2124 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2124-2124 ◽

Cited By ~ 1

Author(s):

Hua Jiang ◽

Gang An ◽

Chirag Acharya ◽

Mike Y Zhong ◽

Ti Cai ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Death ◽

Cell Lines ◽

Caspase 3 ◽

Single Agent ◽

Annexin V ◽

Cell Killing ◽

Individual Agent ◽

Direct Cell ◽

Direct Killing

Abstract SAR650984 (SAR) is a naked humanized IgG1 monoclonal antibody (mAb) selectively targeting the membrane protein CD38 in early clinical development to treat multiple myeloma (MM) and other CD38+ hematological malignancies. SAR has demonstrated encouraging single agent activity in relapsed/refractory (R/R) MM patients (ASCO abstract #8532) and even better efficacy when combined with Dexamethasone and Lenalidomide (Len), without reaching a maximum tolerated dose in patients with heavily pretreated MM (ASCO Abstract #8512). It functions through multiple mechanisms including antibody dependent cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), and direct killing against CD38-positive tumor cells including MM. Although SAR induces lysis of all CD38-expressing MM cell lines via ADCC, it only significantly induces direct killing of MOLP8 cells that express the highest CD38 surface density (~580,000/cell) among > 17 MM cell lines. We first sought to determine whether direct cell death induced by SAR depends on CD38 levels on MM cell membrane by generating RPMI8226 cells overexpressing CD38 (R-CD38) (Abstract #67338). R-CD38 cells express > 6-fold higher CD38 mRNA and surface protein levels than parental RPMI8226 cells (577,304/cell vs. 128,713/cell). Direct MM cell killing by SAR was determined using caspase 3/7 activity and CellTiter-Glo luminescent cell viability assays without goat anti-human IgG crosslinking, in the presence or absence of IL-6 or bone marrow stromal cells (BMSCs). Following overnight incubation, SAR significantly induced homotypic aggregation (HA) of R-CD38, but not control RPMI8226 cells, associated with dose-dependent activation of pro-apoptotic caspase 3/7 in R-CD38, but not control cells. Importantly, SAR decreased the viability of R-CD38, but not control cells, regardless of the presence of IL-6 or BMSCs. Direct cell death induced by SAR depends on SAR-induced HA in MM cells since SAR only blocked survival of R-CD38 and MOLP8 MM cells that show significant HA. Thus, direct apoptosis induced by SAR depends on the level of CD38 surface expression, which may contribute to clinical responses in R/R MM expressing higher CD38 levels. Next, we evaluated the combination effect of Len or Pomalidomide (Pom) with SAR on MM cells. BM mononuclear cells from MM patients were incubated with SAR (10 mg/ml) with or without 10 mM of Len or Pom overnight, followed by flow cytometric analysis to determine % Annexin V/PI staining of CD138+/BCMA+ MM cells. As expected, Pom alone induced slightly higher % of Annexin V+/PI+ MM cells than Len (41 + 1.8 % vs 49 + 1.5 %). Either combination further increased the % of double positive MM patient cells when compared with individual agent alone (from 40 + 2.1% to 70 + 3.1% combined with Len; from 40 + 2.1% to 86 + 3.4% combined with Pom). In addition, PBMC effectors from normal donors (n=4) were pretreated with Len or Pom (5 mM) for 3-7 days and used for SAR-mediated ADCC assays against MM cells (MM1S, MM1R, RPMI8226, R-38, MOLP8), with or without HS-5 or BMSCs from patients. Pom, more potently than Len, further increased SAR-induced MM cell lysis regardless of the presence of BMSCs. Moreover, additional pretreatment of MM cells with Pom overnight further enhanced SAR-induced ADCC by Pom-pretreated PBMC effectors. Both MOLP8 and R-CD38 are relative resistant to direct cytotoxicities induced by Len or Pom. Significantly, Pom, also more potently than Len, augmented direct toxicities induced by SAR in MOLP8 and R-CD38 MM cells. Taken together, we here demonstrate that SAR directly induces apoptosis of MM cells with higher CD38 levels; and that Pom, more effectively than Len, increases SAR-induced MM cell killing via apoptosis and ADCC. These data strongly support SAR as a monotherapy or in combination treatment to improve the outcome of MM patients. Disclosures Cai: Sanofi: Employment. Song:Sanofi: Employment. Yang:Sanofi: Employment. Adrian:Sanofi: Employment. Munshi:Celgene: Consultancy; Onyx: Consultancy; Janssen: Consultancy; Sanofi-Aventis: Consultancy; Ocopep: Consultancy, Equity Ownership, Patents & Royalties. Anderson:Celgene: Consultancy; Onyx: Consultancy; Gilead Sciences: Consultancy; Sanofi-Aventis US: Consultancy; Acetylon: Scientific Founder Other; Oncoprep: Scientific Founder Other.

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Preclinical studies evaluate pivotal TNFα nanomedicine clinical trial design.

Journal of Clinical Oncology ◽

10.1200/jco.2019.37.15_suppl.e14646 ◽

2019 ◽

Vol 37 (15_suppl) ◽

pp. e14646-e14646

Author(s):

Naris Nilubol ◽

David Oarr ◽

Lawrence Tamarkin

Keyword(s):

Clinical Trial ◽

Cancer Cells ◽

Cancer Patients ◽

Cell Lines ◽

Cancer Cell ◽

Single Agent ◽

Fluid Pressure ◽

Standard Of Care ◽

Preclinical Studies ◽

Preclinical Research

e14646 Background: The clinically-tested CYT-6091 nanomedicine is comprised of TNF alpha (TNF) bound to 27 nm pegylated gold nanoparticles. Preclinical research has shown that CYT-6091 disrupts solid tumor blood vessels, reduces interstitial fluid pressure and increases accumulation of follow-on chemotherapy in the tumor microenvironment. While systemic toxicity has limited treatment of cancer patients with TNF, isolated limb perfusion of patients with in-transit melanoma using 1-4 mg of TNF followed by chemotherapy has demonstrated up to 90% complete response rates with one treatment. In a single-agent, phase I clinical study in advanced-stage cancer patients, we found that multiple CYT-6091 treatments can systemically and safely deliver up to 1.2 mg of TNF per dose. Methods: Our current research was conducted to validate administering CYT-6091 prior to standard-of-care chemotherapy in a phase II clinical trial. As part of the NCI’s Experimental Therapeutics (NExT) evaluation, CYT-6091 was studied in combination with paclitaxel (PTX) in C57BL/6 murine xenografts, using three cancer cell lines: MC-38 colon carcinoma (cancer cells sensitive to TNF, but insensitive to PTX), B16/F10 melanoma (cancer cells insensitive to TNF, but sensitive to PTX), Lewis lung carcinoma (cancer cells insensitive to both TNF and PTX), where sensitivity was based on inhibiting cancer cell growth at the studied dosing amounts and frequency. CYT-6091 was administered 3-hours before PTX, and three PTX doses were administered in each model. Results: In every model, CYT-6091 + any dose of PTX was more effective in reducing or controlling tumor growth than the highest dose of PTX alone (p < 0.01). Interestingly, we also found that CYT-6091 alone demonstrated significant anti-cancer activity (p < 0.01), suggesting that additional studies may be warranted to determine optimal frequency and dose of CYT-6091 as a single agent. Conclusions: These preclinical studies demonstrate for the first time that CYT-6091 + PTX was superior to PTX (p < 0.01) or CYT-6091 alone (P < 0.05) in TNF insensitive cell lines, which represent the majority of cancers. Findings support systemically administering CYT-6091 plus standard-of-care chemotherapy in a pivotal clinical trial.

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The Novel Triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic Acid (CDDO) Induces Apoptosis of Human Diffuse Large B-Cell Lymphoma Cells (DLCL) through a Peroxisome Proliferator-Activated Receptor γ (PPARγ) Independent Pathway Which Is Enhanced by NFκB Inhibition.

Blood ◽

10.1182/blood.v106.11.2425.2425 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2425-2425

Author(s):

Denise Ray ◽

Kimberly Morse ◽

Shannon Hilchey ◽

Tatiana Garcia ◽

Raymond Felgar ◽

...

Keyword(s):

Clinical Trials ◽

Cell Death ◽

B Cell ◽

Transcriptional Activity ◽

Nuclear Translocation ◽

Single Agent ◽

B Cell Lymphoma ◽

Cell Phenotype ◽

Clinical Activity ◽

Peroxisome Proliferator

Abstract Ligands for the transcription factor PPARγ are emerging as a new class of anti-tumor agents. Herein we report that the synthetic triterpenoid CDDO, a PPARγ ligand that induces PPARγ transcriptional activity in human DLCL OCI-Ly-19 cells, also induces cell death in human DLCL of both germinal center (OCI-Ly19) and activated B-cell phenotype (OCI-Ly10), cells which express the PPARγ protein. This effect of CDDO appears to be independent of PPARγ stimulated pathways since the functional antagonist of PPARγ, GW9662, which completely inhibits CDDO induced PPARγ transcriptional activity was unable to prevent CDDO induced cell death. Similar findings were seen using the additional PPARγ antagonists T0070907 and BADGE. CDDO induces cell death by inhibiting cell proliferation and inducing apoptosis as shown by Annexin-V and propidium iodide staining. As we have previously shown that PPARγ ligands inhibit NF-κB activity in B lymphocytes (J. Immunol2005; 174(7): 4060–9), we next examined the effect of CDDO on NF-κB in DLCL cells. Surprisingly, exposure of Ly19 cells to CDDO resulted in a dose-, and time-dependent increase in the activity of both the p50 and p65 subunits of NF-κB as determined by ELISA, by direct visualization of the nuclear translocation of p65 using indirect immunofluorescence assays, and by EMSA. The nuclear translocation of both the p50 and p65 NF-κB subunits was also confirmed by performing immunoblot analyses using nuclear fractions of CDDO-treated Ly19 cells. NF-κB activation was also observed in Ly10 cells exposed to CDDO. Follow-up experiments revealed that the activation of NF-κB in Ly19 cells by CDDO was due to proteolysis of inhibitory IκBα molecules. To determine whether the CDDO-induced NF-κB activation was a pro- survival mechanism, Ly19 and Ly10 cells were pre-treated with the NF-κB inhibitors SN50, helenalin or BAY 11-7082 and then exposed to CDDO for 24 hrs. In all cases, the NF-κB inhibitors significantly enhanced CDDO induced cell death suggesting that NF-κB activation is an anti-apoptotic mechanism elicited to protect the cell against CDDO cytotoxicity. Collectively, these studies suggest that; (a) CDDO (which will shortly be entering clinical trials for patients with acute myeloid leukemia) as a single agent may have significant clinical activity in patients with DLCL and; (b) the combination of CDDO with pharmacological inhibitors of NF-κB would be a rationale combination of novel agents to test in the context of clinical trials for patients with DLCL.

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