Forging a new path with cyclin-dependent kinase inhibitors to treat epithelioid hemangioendothelioma

PROJECT SUMMARY Epithelioid hemangioendothelioma (EHE) is a rare monogenic cancer caused by a reciprocal chromosomal translocation that generates the TAZ(WWTR1)–CAMTA1 (TC) fusion gene. No effective treatments exist for patients with aggressive EHE who do not survive beyond 2-years post-diagnosis. Further, no human cell models

of EHE exist, making both preclinical testing of potential therapeutics and a mechanistic understanding of aggressive disease quite challenging. To begin to address these problems and identify potential treatments, we generated two genetically engineered mouse models (GEMM) of EHE: an indolent/less aggressive model, by

recreating TC fusion at the endogenous Taz locus, and an aggressive EHE model by combining TC fusion with a cyclin-dependent kinase inhibitor 2a (Cdkn2a) conditional knockout. We also derived the first EHE cell lines from aggressive tumors via ex vivo expansion; however, we were unable to develop cell lines using the

indolent/less aggressive model. TC is a transcriptional coregulator that is predominantly localized to the nucleus; therefore, we reasoned that drugs/small molecules that translocate TC to the cytoplasm or destabilize TC would reduce oncogenicity. To this end, we performed phenotypic screening based on TC immunofluorescence to

identify such drugs/small molecules. We identified the cyclin-dependent kinase inhibitor (CDKi) dinaciclib as a potent drug that can cause TC nuclear exit and destabilization. This translates to decreased cell proliferation and enhancement of apoptosis, as EHE cells are ‘oncogene-addicted’ to TC. Our long-term goal is to understand

EHE tumor biology and identify a therapeutic vulnerability for the effective treatment of EHE. Aim 1: We will determine the efficacy of dinaciclib in allograft models of EHE and elucidate its mechanism of action. All the top hits in our phenotypic screen, including dinaciclib, were transcription inhibitors that prevent the assembly of the

productive transcription elongation complex. Therefore, we hypothesize that incorporating TC into the productive elongation complex prevents its nuclear exit. To test this hypothesis, we will evaluate by immunoprecipitation and mass spectrometry whether TC binds to components of this complex and will characterize the TC

interactome after treatment with transcription inhibitors. Aim 2: Evaluate the effect of Cdkn2a introduction on TC localization and stability in vitro and in allograft models by introducing p16Ink4a and p19Arf, the two major protein products of Cdkn2a, into our EHE cell line via CRISPR-HDR. Given that CDK inhibition destabilizes TC, we

hypothesize that Cdkn2a, a physiological CDKi, will destabilize TC. Clinically, CDKN2A loss is associated with late-stage or aggressive disease; however, the underlying molecular mechanisms remain unknown. Therefore, we will investigate if Cdkn2a decreases TC stability. In sum, this work will be significant because we will have

shown that dinaciclib is potentially the first effective treatment for aggressive EHE, and we will have deciphered the mechanism linking CDK inhibition and TC nuclear exit/stability.