Neratinib inhibits Hippo/YAP signaling, reduces mutant K-RAS expression, and kills pancreatic and blood cancer cells

Dent, Paul; Booth, Laurence; Roberts, Jeanette C.; Liu, Junchen; Poklepovic, Andrew; Lalani, Alshad S.; Tuveson, David A.; Martinez, Jennifer; Hancock, John F.

doi:10.1038/s41388-019-0849-8

Cited by 68 publications

(130 citation statements)

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“…in agreement with prior data in pancreatic cancer cells, Jurkat T cells, and HL60 APL cells, it profoundly reduced Ezrin T567 phosphorylation in the cutaneous T cells ( Figure 5) (Dent, Booth, Roberts et al, 2019). What was most noticeable at 60× magnification were the filamentous projections extending beyond the plasma membrane of the T cells, where phospho-Ezrin exhibited a punctate zebra-like staining pattern.…”

supporting

confidence: 92%

Neratinib degrades MST4 via autophagy that reduces membrane stiffness and is essential for the inactivation of PI3K, ERK1/2, and YAP/TAZ signaling

Dent

Booth

Poklepovic

et al. 2020

Journal Cellular Physiology

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The irreversible ERBB1/2/4 inhibitor neratinib causes plasma membrane‐associated K‐RAS to mislocalize into intracellular vesicles liminal to the plasma membrane; this effect is enhanced by HDAC inhibitors and is now a Phase I trial (NCT03919292). The combination of neratinib and HDAC inhibitors killed pancreatic cancer and lymphoma T cells. Neratinib plus HDAC inhibitor exposure was as efficacious as (paclitaxel+gemcitabine) at killing pancreatic cancer cells. Neratinib reduced the phosphorylation of PAK1, Merlin, LATS1/2, AKT, mTOR, p70 S6K, and ERK1/2 which required expression of Rubicon, Beclin1, and Merlin. Neratinib altered pancreatic tumor cell morphology which was associated with MST4 degradation reduced Ezrin phosphorylation and enhanced phosphorylation of MAP4K4 and LATS1/2. Knockdown of the MAP4K4 activator and sensor of membrane rigidity RAP2A reduced basal LATS1/2 and YAP phosphorylation but did not prevent neratinib from stimulating LATS1/2 or YAP phosphorylation. Beclin1 knockdown prevented MST4 degradation, Ezrin dephosphorylation and neratinib‐induced alterations in tumor cell morphology. Our findings demonstrate that neratinib enhances LATS1/2 phosphorylation independently of RAP2A/MAP4K4 and that MST4 degradation and Ezrin dephosphorylation may represent a universal trigger for the biological actions of neratinib.

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confidence: 92%

Neratinib degrades MST4 via autophagy that reduces membrane stiffness and is essential for the inactivation of PI3K, ERK1/2, and YAP/TAZ signaling

Dent

Booth

Poklepovic

et al. 2020

Journal Cellular Physiology

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“…As part of the induction of autophagy, HDAC protein expression was reduced which correlated with enhanced expression of Class I MHCA and reduced expression of PD‐L1. Whether plasma membrane proteins such as PD‐L1 are being degraded via LC3‐associated phagocytosis will require studies beyond this manuscript (Dent, Booth, Roberts, et al, 2020; Dent, Booth, Roberts, Liu, et al, 2019). Using transient molecular knockdown approaches, HDACs whose expression was reduced via autophagy, that is HDACs1/2/3, were shown to be mechanistically responsible for the changes in MHCA and PD‐L1 expression.…”

Section: Discussionmentioning

confidence: 99%

GZ17‐6.02 initiates DNA damage causing autophagosome‐dependent HDAC degradation resulting in enhanced anti‐PD1 checkpoint inhibitory antibody efficacy

Booth

Roberts

West

et al. 2020

Journal Cellular Physiology

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Our studies examined the molecular mechanisms by which the novel cancer therapeutic GZ17‐6.02 (NCT03775525) killed GI tumor cells. TZ17‐6.02 activated ATM which was responsible for increased phosphorylation of nuclear γH2AX and AMPKα T172. ATM‐AMPK signaling was responsible for the subsequent inactivation of mTORC1 and mTORC2, dephosphorylation of ULK1 S757, and increased phosphorylation of ULK1 S317 and of ATG13 S318, which collectively caused enhanced autophagosome formation. GZ17‐6.02 interacted with 5‐fluorouracil in an additive to greater than additive fashion to kill all of the tested GI tumor cell types. This was associated with greater ATM activation and a greater mammalian target of rapamycin inactivation and autophagosome induction. As a result, autophagy‐dependent degradation of multiple histone deacetylase (HDAC) proteins and chaperone proteins occurred. Loss of HDAC expression was causal in reduced expression of programed death ligand 1 (PD‐L1), ornithine decarboxylase, and indole amine 2,3‐dioxygenase (IDO1) and in the elevated expression of major histocompatibility complex Class IA (MHCA). Treatment with GZ17‐6.02 also resulted in enhanced efficacy of a subsequently administered anti‐PD1 checkpoint inhibitory antibody. Thus, the primary mode of GZ17‐6.02 action is to induce a DNA damage response concomitant with ATM activation, that triggers a series of interconnected molecular events that result in tumor cell death and enhanced immunogenicity.

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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: 86%

“…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%

“…We have recently demonstrated that the irreversible ERBB1/2/4 inhibitor neratinib could down-regulate the expression of RAS proteins, and based on this data and the fact that phosphorylated fingolimod causes sphingosine-1-phosphate receptor 1 internalization and degradation, we wished to determine whether this drug could act upon RAS proteins in GBM cells, in a manner similar to neratinib (20,21,24,31). In GBM6 cells, that express an NH2-terminal truncated active ERBB1 vIII [fingolimod + MMF], caused intracellular clustering of the receptor; unlike fingolimod, our positive control neratinib increased clustering followed later by reduced expression of the receptor (Figure 3A).…”

Section: Resultsmentioning

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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Neratinib inhibits Hippo/YAP signaling, reduces mutant K-RAS expression, and kills pancreatic and blood cancer cells

Cited by 68 publications

References 50 publications

Neratinib degrades MST4 via autophagy that reduces membrane stiffness and is essential for the inactivation of PI3K, ERK1/2, and YAP/TAZ signaling

Neratinib degrades MST4 via autophagy that reduces membrane stiffness and is essential for the inactivation of PI3K, ERK1/2, and YAP/TAZ signaling

GZ17‐6.02 initiates DNA damage causing autophagosome‐dependent HDAC degradation resulting in enhanced anti‐PD1 checkpoint inhibitory antibody efficacy

Fingolimod Augments Monomethylfumarate Killing of GBM Cells

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