HDAC inhibitors enhance neratinib activity and when combined enhance the actions of an anti-PD-1 immunomodulatory antibody <i>in vivo</i>

Booth, Laurence; Roberts, Jeanette C.; Poklepovic, Andrew; Avogadri-Connors, Francesca; Cutler, Richard E.; Lalani, Alshad S.; Dent, Paul

doi:10.18632/oncotarget.21660

Cited by 57 publications

(153 citation statements)

References 43 publications

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“…In GBM6, but not GBM14, the drugs combined to further activate ATM and AMPK; in several prior studies using different drug combinations we have delineated an ATM-AMPK-ULK1-ATG13 pathway that promotes autophagosome formation (Figures 2A,B) (20,21,24,31). In GBM14 cells, fingolimod as a single agent strongly activated ATM-AMPK signaling.…”

Section: Resultsmentioning

confidence: 85%

“…In many prior experimental therapeutics studies, we have performed agnostic screening analyses following exposure of cancer cells to drugs using the Hermes widefield microscope. Using fluorescence intensity imaging of individual cells, we define the changes in the expression and phosphorylation of multiple signal transduction proteins (20,21,24,31). Signaling by ERK1/2, AKT, mTOR, p70 S6K, STAT3, and STAT5 was assessed, as was signaling by ATM, the AMPK and eIF2 alpha.…”

Section: Resultsmentioning

confidence: 99%

“…Trypsin-EDTA, DMEM, RPMI, penicillin-streptomycin were purchased from GIBCOBRL (GIBCOBRL Life Technologies, Grand Island, NY). Other reagents and performance of experimental procedures were as described (15,(20)(21)(22)(23)(24). Antibodies used: AIF (5318), BAX (5023), BAK (12105), BAD (9239), BIM (2933), BAK1 (12105), Beclin1 (3495), cathepsin B (31718), CD95 (8023), FADD (2782), eIF2α (5324), P-eIF2α S51 (3398), ULK-1 (8054), P-ULK-1 S757 (14202), P-AMPK S51 (2535), AMPKα (2532), P-ATM S1981 (13050), ATM (2873), ATG5 (12994), mTOR (2983), P-mTOR S2448 (5536), P-mTOR S2481 (2974), ATG13 (13468), MCL-1 (94296), BCL-XL (2764), P-AKT T308 (13038), P-ERK1/2 (5726), P-STAT3 Y705 (9145), P-p65 S536 (3033), p62 (23214), LAMP2 (49067) all from Cell Signaling Technology; P-ULK-1 S317 (3803a) from Abgent; P-ATG13 S318 (19127) from Novus Biologicals.…”

Section: Methodsmentioning

confidence: 99%

“…Freshly isolated GBM cells and activated microglia directly from the operating room were separated and grown in RPMI supplemented with 2.0% (v/v) fetal calf serum and 10% (v/v) Non-essential amino acids for 6 h, followed by drug exposure and viability assessments made the following day (15,(25)(26)(27). Cells were transfected with siRNA molecules or plasmids as described in prior manuscripts (20)(21)(22)(23)(24). Cells were transfected with a plasmid to express GFP-K-RAS V12 (0.1 µg) using lipofectamine 2000.…”

Section: Culture Transfection and In Vitro Exposure Of Cells To Drugsmentioning

confidence: 99%

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Fingolimod Augments Monomethylfumarate Killing of GBM Cells

et al. 2020

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Previously we demonstrated that the multiple sclerosis drug dimethyl fumarate (DMF) and its plasma breakdown product MMF could interact with chemotherapeutic agents to kill both GBM cells and activated microglia. The trial NCT02337426 demonstrated the safety of DMF in newly diagnosed GBM patients when combined with the standard of care Stupp protocol. We hypothesized that another multiple sclerosis drug, fingolimod (FTY720) would synergize with MMF to kill GBM cells. MMF and fingolimod interacted in a greater than additive fashion to kill PDX GBM isolates. MMF and fingolimod radiosensitized glioma cells and enhanced the lethality of temozolomide. Exposure to [MMF + fingolimod] activated an ATM-dependent toxic autophagy pathway, enhanced protective endoplasmic reticulum stress signaling, and inactivated protective PI3K, STAT, and YAP function. The drug combination reduced the expression of protective c-FLIP-s, MCL-1, BCL-XL, and in parallel caused cell-surface clustering of the death receptor CD95. Knock down of CD95 or over-expression of c-FLIP-s or BCL-XL suppressed killing. Fingolimod and MMF interacted in a greater than additive fashion to rapidly enhance reactive oxygen species production and over-expression of either thioredoxin or super-oxide dismutase two significantly reduced the drug-induced phosphorylation of ATM, autophagosome formation and [MMF + fingolimod] lethality. In contrast, the production of ROS was only marginally reduced in cells lacking ATM, CD95, or Beclin1. Collectively, our data demonstrate that the primary generation of ROS by [MMF + fingolimod] plays a key role, via the induction of toxic autophagy and death receptor signaling, in the killing of GBM cells.

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Section: Resultsmentioning

confidence: 85%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Culture Transfection and In Vitro Exposure Of Cells To Drugsmentioning

confidence: 99%

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Fingolimod Augments Monomethylfumarate Killing of GBM Cells

et al. 2020

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“…It also has been demonstrated that programmed cell death 1 or PD‐L1 expression was increased, supporting a role for combining HDAC inhibitors with immune checkpoint inhibitors (Fig. ) . Table lists representative clinical trials of these types of drug combinations in solid tumors actively enrolling as of June 2018.…”

Section: Examples Of Recruiting Clinical Trials Using Combinations Ofmentioning

confidence: 84%

Entinostat finds a path: A new study elucidates effects of the histone deacetylase inhibitor on the immune system

Surolia

Bates

2018

Cancer

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Epigenetic therapies have acquired a solid place in clinical armamentariums with the recent approval of enasidenib for isocitrate dehydrogenase 2 (IDH2)-mutated acute myeloid leukemias. This adds to previous approvals of 4 histone deacetylase (HDAC) inhibitors for T-cell lymphomas or multiple myeloma in addition to the long-approved demethylating agents for myeloid disease. But, frustratingly, solid tumor indications have not emerged despite significant efforts, including numerous clinical trials with combination therapies. One recent strategy that has emerged bears perhaps more promise: the use of HDAC inhibitors and DNA-demethylating agents to upregulate components of the immune response and thereby enhance the efficacy of immunotherapy agents, particularly the immune checkpoint blockers. In this issue of Cancer, Smith and colleagues 1 demonstrate the antitumor activity of entinostat in a murine model of ovarian cancer and indicate that efficacy requires adaptive immunity. Such a finding adds support to the ongoing combination studies with immune checkpoint blockade and suggests that this strategy could be applied in ovarian cancer.HDAC inhibitors are epigenetic agents that have been studied intensively for their ability to induce histone hyperacetylation and to upregulate the expression of genes of differentiation and cell cycle control. They do this by inhibiting the HDAC-mediated enzymatic removal of acetyl groups on chromatin, resulting in the aberrant silencing of important tumor suppressors in cancer. Entinostat follows a long history of drug development that began with butyric acid derivatives in the 1980s. Butyrates were the first agents for which histone hyperacetylation was linked with differentiation-eg, with the induction of hemoglobin synthesis in K562 erythroleukemia cells. 2 Searches for other differentiating agents led to identification of the hydroxamic acid vorinostat, and its ability to inhibit HDACs was reported in 1998. 3,4 . Romidepsin was identified for its ability target Ras; but, likewise its ability to inhibit HDACs was reported in 1998. 5-7 Clinical activity and US Food and Drug Administration approval for vorinostat in 2006 and romidepsin in 2009 triggered the development of a spate of HDAC inhibitors, most of them hydroxamic acids like vorinostat. 8 Two other hydroxamic acids gained US Food and Drug Administration approval: belinostat for peripheral T-cell lymphoma and panobinostat in combination with bortezomib and dexamethasone for multiple myeloma. A different drug class was added to the HDAC inhibitors, the benzamides, including entinostat/MS275 and mocetinostat, 9 and, later, chidamide. The first clinical trials of entinostat were reported in solid tumors, leukemias, and lymphomas in 2006 and 2007. 10,11 The enzyme targets for these are the HDAC enzymes, which are divided by structure into 4 classes: enzymes 1, 2, 3, and 8 comprise class I; enzymes 4, 5, 7, and 9 comprise class IIA; enzymes 6 and 10 comprise class IIB; and HDAC11 constitutes class IV. 12 The enzymes are struc...

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Neratinib augments the lethality of [regorafenib + sildenafil]

Booth

Roberts

Rais

et al. 2018

Journal Cellular Physiology

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Regorafenib is approved for the treatment of colorectal cancer and hepatocellular carcinoma. In the trial NCT02466802, we have discovered that regorafenib can be safely combined with the phosphodiesterase 5 inhibitor sildenafil in advanced solid tumor patients. The present studies determined whether the approved ERBB1/2/4 and RAS downregulating drug neratinib, could enhance the lethality of [regorafenib + sildenafil]. Neratinib enhanced [regorafenib + sildenafil] lethality in a greater than additive fashion in colon cancer cells. The drug combination reduced the expression of mutant K-RAS and of multiple histone deacetylase (HDAC) proteins that required autophagosome formation. It caused green fluorescent protein or red fluorescent protein-tagged forms of K-RAS V12 to localize into large intracellular vesicles. Compared with [regorafenib + sildenafil], the three-drug combination caused greater and more prolonged activation of the ATM-AMPK-ULK-1 pathway and caused a greater suppression and prolonged inactivation of mammalian target of rapamycin, AKT, and p70 S6K. Approximately 70% of enhanced lethality caused by neratinib required ataxia-telangiectasia-mutated (ATM)-AMP-dependent protein kinase (AMPK) signaling whereas knockdown of Beclin1, ATG5, FADD, and CD95 completely prevented the elevated killing effect. Exposure of cells to [regorafenib + sildenafil] reduced the expression of the checkpoint immunotherapy biomarkers programmed death-ligand 1, ornithine decarboxylase, and indoleamine 2,3-dioxygenase-1 and increased the expression of major histocompatibility complex A (MHCA), which also required autophagosome formation. Knockdown of specific HDAC proteins recapitulated the effects observed using chemical agents. In vivo, using mouse cancer models, neratinib significantly enhanced the antitumor efficacy of [regorafenib + sildenafil]. Our data support performing a new three drug Phase I trial combining regorafenib, sildenafil, and neratinib.

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HDAC inhibitors enhance neratinib activity and when combined enhance the actions of an anti-PD-1 immunomodulatory antibody in vivo

Cited by 57 publications

References 43 publications

Fingolimod Augments Monomethylfumarate Killing of GBM Cells

Fingolimod Augments Monomethylfumarate Killing of GBM Cells

Entinostat finds a path: A new study elucidates effects of the histone deacetylase inhibitor on the immune system

Neratinib augments the lethality of [regorafenib + sildenafil]

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