ABSTRACT
Targeting surface receptors overexpressed on cancer cells is one way to specifically treat cancer versus normal cells. Vintafolide (EC145), which consists of folate linked to a cytotoxic small molecule, desacetylvinblastine hydrazide (DAVLBH), takes advantage of the overexpression of folate receptor (FR) on cancer cells. Once bound to FR, vintafolide enters the cell by endocytosis, and the reducing environment of the endosome cleaves the linker, releasing DAVLBH to destabilize microtubules. Vintafolide has shown efficacy and improved tolerability compared with DAVLBH in FR-positive preclinical models. As the first FR-targeting drug to reach the clinic, vintafolide has achieved favorable responses in phase II clinical trials in FR-positive ovarian and lung cancer. However, some FR-positive patients in these clinical trials do not respond to vintafolide. We sought to identify potential biomarkers of resistance to aid in the future development of this and other FR-targeting drugs. Here, we confirm that high P-glycoprotein (P-gp) expression was the strongest predictor of resistance to DAVLBH in a panel of 359 cancer cell lines. Furthermore, targeted delivery of DAVLBH via the FR, as in vintafolide, fails to overcome P-gp-mediated efflux of DAVLBH in both in vitro and in vivo preclinical models. Therefore, we suggest that patients whose tumors express high levels of P-gp be excluded from future clinical trials for vintafolide as well as other FR-targeted therapeutics bearing a P-gp substrate. Mol Cancer Ther; 15(8); 1998-2008. ©2016 AACR.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B/genetics , Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm/genetics , Folic Acid/analogs & derivatives , Gene Expression , Vinca Alkaloids/pharmacology , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/genetics , Cluster Analysis , Computational Biology/methods , Disease Models, Animal , Dose-Response Relationship, Drug , Female , Folate Receptors, GPI-Anchored/antagonists & inhibitors , Folic Acid/pharmacology , Gene Expression Profiling , Humans , Mice , Platinum/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Inhibition of the DNA damage checkpoint kinase WEE1 potentiates genotoxic chemotherapies by abrogating cell-cycle arrest and proper DNA repair. However, WEE1 is also essential for unperturbed cell division in the absence of extrinsic insult. Here, we investigate the anticancer potential of a WEE1 inhibitor, independent of chemotherapy, and explore a possible cellular context underlying sensitivity to WEE1 inhibition. We show that MK-1775, a potent and selective ATP-competitive inhibitor of WEE1, is cytotoxic across a broad panel of tumor cell lines and induces DNA double-strand breaks. MK-1775-induced DNA damage occurs without added chemotherapy or radiation in S-phase cells and relies on active DNA replication. At tolerated doses, MK-1775 treatment leads to xenograft tumor growth inhibition or regression. To begin addressing potential response markers for MK-1775 monotherapy, we focused on PKMYT1, a kinase functionally related to WEE1. Knockdown of PKMYT1 lowers the EC(50) of MK-1775 by five-fold but has no effect on the cell-based response to other cytotoxic drugs. In addition, knockdown of PKMYT1 increases markers of DNA damage, γH2AX and pCHK1(S345), induced by MK-1775. In a post hoc analysis of 305 cell lines treated with MK-1775, we found that expression of PKMYT1 was below average in 73% of the 33 most sensitive cell lines. Our findings provide rationale for WEE1 inhibition as a potent anticancer therapy independent of a genotoxic partner and suggest that low PKMYT1 expression could serve as an enrichment biomarker for MK-1775 sensitivity.
Subject(s)
Antineoplastic Agents/pharmacology , Cell Cycle Proteins/antagonists & inhibitors , Nuclear Proteins/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/chemistry , Cell Cycle/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , DNA Damage/drug effects , Disease Models, Animal , Drug Evaluation, Preclinical , Drug Resistance, Neoplasm/genetics , Female , Gene Knockdown Techniques , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/chemistry , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Pyrazoles/administration & dosage , Pyrimidines/administration & dosage , Pyrimidinones , Tumor Burden/drug effects , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: Inhibition of kinases involved in the DNA damage response sensitizes cells to genotoxic agents by abrogating checkpoint-induced cell cycle arrest. CHK1 and WEE1 act in a pathway upstream of CDK1 to inhibit cell cycle progression in response to damaged DNA. Therapeutic targeting of either CHK1 or WEE1, in combination with chemotherapy, is under clinical evaluation. These studies examine the overlap and potential for synergy when CHK1 and WEE1 are inhibited in cancer cell models. METHODS: Small molecules MK-8776 and MK-1775 were used to selectively and potently inhibit CHK1 and WEE1, respectively. RESULTS: In vitro, the combination of MK-8776 and MK-1775 induces up to 50-fold more DNA damage than either MK-8776 or MK-1775 alone at a fixed concentration. This requires aberrant cyclin-dependent kinase activity but does not appear to be dependent on p53 status alone. Furthermore, DNA damage takes place primarily in S-phase cells, implying disrupted DNA replication. When dosed together, the combination of MK-8776 and MK-1775 induced more intense and more durable DNA damage as well as anti-tumor efficacy than either MK-8776 or MK-1775 dosed alone. DNA damage induced by the combination was detected in up to 40% of cells in a treated xenograft tumor model. CONCLUSIONS: These results highlight the roles of WEE1 and CHK1 in maintaining genomic integrity. Importantly, the strong synergy observed upon inhibition of both kinases suggests unique yet complimentary anti-tumor effects of WEE1 and CHK1 inhibition. This demonstration of DNA double strand breaks in the absence of a DNA damaging chemotherapeutic provides preclinical rationale for combining WEE1 and CHK1 inhibitors as a cancer treatment regimen.
ABSTRACT
How do myelinated axons signal to the nuclei of cells that enwrap them? The cell bodies of oligodendrocytes and Schwann cells are segregated from axons by multiple layers of bimolecular lipid leaflet and myelin proteins. Conventional signal transduction strategies would seem inadequate to the challenge without special adaptations. Wallerian degeneration provides a model to study axon-to-Schwann cell signaling in the context of nerve injury. We show a hitherto undetected rapid, but transient, activation of the receptor tyrosine kinase erbB2 in myelinating Schwann cells after sciatic nerve axotomy. Deconvolving microscopy using phosphorylation state-specific antibodies shows that erbB2 activation emanates from within the microvilli of Schwann cells, in direct contact with the axons they enwrap. To define the functional role of this transient activation, we used a small molecule antagonist of erbB2 activation (PKI166). The response of myelinating Schwann cells to axotomy is inhibited by PKI166 in vivo. Using neuron/Schwann cell cocultures prepared in compartmentalized cell culture chambers, we show that even transient activation of erbB2 is sufficient to initiate Schwann cell demyelination and that the initiating functions of erbB2 are localized to Schwann cells.
