Sheryl M. Gough

Structural chromosomal rearrangements of the Nucleoporin 98 gene (NUP98), primarily balanced translocations and inversions, are associated with a wide array of hematopoietic malignancies. NUP98 is known to be fused to at least 28 different partner genes in patients with hematopoietic malignancies, including acute myeloid leukemia, chronic myeloid leukemia in blast crisis, myelodysplastic syndrome, acute lymphoblastic leukemia, and bilineage/biphenotypic leukemia. NUP98 gene fusions typically encode a fusion protein that retains the amino terminus of NUP98; in this context, it is important to note that several recent studies have demonstrated that the amino-terminal portion of NUP98 exhibits transcription activation potential. Approximately half of the NUP98 fusion partners encode homeodomain proteins, and at least 5 NUP98 fusions involve known histone-modifying genes. Several of the NUP98 fusions, including NUP98-homeobox (HOX)A9, NUP98-HOXD13, and NUP98-JARID1A, have been used to generate animal models of both lymphoid and myeloid malignancy; these models typically up-regulate HOXA cluster genes, including HOXA5, HOXA7, HOXA9, and HOXA10. In addition, several of the NUP98 fusion proteins have been shown to inhibit differentiation of hematopoietic precursors and to increase self-renewal of hematopoietic stem or progenitor cells, providing a potential mechanism for malignant transformation.

PURPOSE: Estrogen receptor (ER) alpha signaling is a known driver of ER-positive (ER+)/human epidermal growth factor receptor 2 negative (HER2-) breast cancer. Combining endocrine therapy (ET) such as fulvestrant with CDK4/6, mTOR, or PI3K inhibitors has become a central strategy in the treatment of ER+ advanced breast cancer. However, suboptimal ER inhibition and resistance resulting from the ESR1 mutation dictates that new therapies are needed. EXPERIMENTAL DESIGN: A medicinal chemistry campaign identified vepdegestrant (ARV-471), a selective, orally bioavailable, and potent small molecule PROteolysis-TArgeting Chimera (PROTAC) degrader of ER. We used biochemical and intracellular target engagement assays to demonstrate the mechanism of action of vepdegestrant, and ESR1 wild-type (WT) and mutant ER+ preclinical breast cancer models to demonstrate ER degradation-mediated tumor growth inhibition (TGI). RESULTS: Vepdegestrant induced ≥90% degradation of wild-type and mutant ER, inhibited ER-dependent breast cancer cell line proliferation in vitro, and achieved substantial TGI (87%-123%) in MCF7 orthotopic xenograft models, better than those of the ET agent fulvestrant (31%-80% TGI). In the hormone independent (HI) mutant ER Y537S patient-derived xenograft (PDX) breast cancer model ST941/HI, vepdegestrant achieved tumor regression and was similarly efficacious in the ST941/HI/PBR palbociclib-resistant model (102% TGI). Vepdegestrant-induced robust tumor regressions in combination with each of the CDK4/6 inhibitors palbociclib, abemaciclib, and ribociclib; the mTOR inhibitor everolimus; and the PI3K inhibitors alpelisib and inavolisib. CONCLUSIONS: Vepdegestrant achieved greater ER degradation in vivo compared with fulvestrant, which correlated with improved TGI, suggesting vepdegestrant could be a more effective backbone ET for patients with ER+/HER2- breast cancer.

Abstract ARV-471, an estrogen receptor (ER) alpha PROTAC® protein degrader, is a hetero-bifunctional molecule that facilitates the interactions between estrogen receptor alpha and an intracellular E3 ligase complex, leading to the ubiquitylation and subsequent degradation of estrogen receptors via the proteasome. ARV-471 robustly degrades ER in ER-positive breast cancer cell lines with a half-maximal degradation concentration (DC50) of ~ 1 nM. PROTAC® mediated ER degradation decreases the expression of classically regulated ER-target genes and inhibits cell proliferation of ER-dependent cell lines (MCF7, T47D). Additionally, ARV-471 degrades clinically relevant ESR1 variants (Y537S and D538G) and inhibits growth of cell lines expressing those variants. In an immature rat uterotrophic model, ARV-471 degrades rat uterine ER and demonstrates no agonist activity. Daily, oral-administration of single agent ARV-471 (3, 10, and 30 mpk) leads to significant anti-tumor activity of estradiol-dependent MCF7 xenografts and concomitant tumor ER protein reductions of >90% at study termination. Moreover, when a CDK4/6 inhibitor is combined with ARV-471 in the MCF7 model, even more pronounced tumor growth inhibition is observed (131% TGI), accompanied by significant reductions in ER protein levels. In an ESR1 Y537S, hormone-independent patient-derived xenograft model, ARV-471 at 10 mpk completely inhibited growth and also significantly reduced mutant ER protein levels. Taken together, the preclinical data of ARV-471 supports its continued development as a best-in-class oral ER PROTAC® protein degrader. These preclinical data supported the clinical development of ARV-471 for the treatment of patients with breast cancer. The discovery, chemical structure and initial clinical data of ARV-471 will be presented. Citation Format: Lawrence B. Snyder, John J. Flanagan, Yimin Qian, Sheryl M. Gough, Monica Andreoli, Mark Bookbinder, Gregory Cadelina, John Bradley, Emma Rousseau, Julian Chandler, Ryan Willard, Jennifer Pizzano, Craig M. Crews, Andrew P. Crew, John Houston, Marcia Dougan Moore, Ron Peck, Ian Taylor. The discovery of ARV-471, an orally bioavailable estrogen receptor degrading PROTAC for the treatment of patients with breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 44.

The myelodysplastic syndrome (MDS) is a clonal hematologic disorder that frequently evolves to acute myeloid leukemia (AML). Its pathogenesis remains unclear, but mutations in epigenetic modifiers are common and the disease often responds to DNA methylation inhibitors. We analyzed DNA methylation in the bone marrow and spleen in two mouse models of MDS/AML, the NUP98-HOXD13 (NHD13) mouse and the RUNX1 mutant mouse model. Methylation array analysis showed an average of 512/3445 (14.9%) genes hypermethylated in NHD13 MDS, and 331 (9.6%) genes hypermethylated in RUNX1 MDS. Thirty-two percent of genes in common between the two models (2/3 NHD13 mice and 2/3 RUNX1 mice) were also hypermethylated in at least two of 19 human MDS samples. Detailed analysis of 41 genes in mice showed progressive drift in DNA methylation from young to old normal bone marrow and spleen; to MDS, where we detected accelerated age-related methylation; and finally to AML, which markedly extends DNA methylation abnormalities. Most of these genes showed similar patterns in human MDS and AML. Repeat element hypomethylation was rare in MDS but marked the transition to AML in some cases. Our data show consistency in patterns of aberrant DNA methylation in human and mouse MDS and suggest that epigenetically, MDS displays an accelerated aging phenotype.

Abstract In this report, we show that expression of a NUP98–PHF23 (NP23) fusion, associated with acute myeloid leukemia (AML) in humans, leads to myeloid, erythroid, T-cell, and B-cell leukemia in mice. The leukemic and preleukemic tissues display a stem cell–like expression signature, including Hoxa, Hoxb, and Meis1 genes. The PHF23 plant homeodomain (PHD) motif is known to bind to H3K4me3 residues, and chromatin immunoprecipitation experiments demonstrated that the NP23 protein binds to chromatin at a specific subset of H3K4me3 sites, including at Hoxa, Hoxb, and Meis1. Treatment of NP23 cells with disulfiram, which inhibits the binding of PHD motifs to H3K4me3, rapidly and selectively killed NP23-expressing myeloblasts; cell death was preceded by decreased expression of Hoxa, Hoxb, and Meis1. Furthermore, AML driven by a related fusion gene, NUP98–JARID1A (NJL), was also sensitive to disulfiram. Thus, the NP23 mouse provides a platform to evaluate compounds that disrupt binding of oncogenic PHD proteins to H3K4me3. Significance: NP23 and NJL belong to a subset of chromatin-modifying fusion oncoproteins that cause leukemia characterized by overexpression of Hoxa and Meis1 genes. Inhibition of NP23 binding to H3K4me3 at Hoxa and Meis1 loci by disulfiram, a U.S. Food and Drug Administration–approved drug, leads to leukemic cell death, demonstrating the feasibility of targeting this subset of oncoproteins. Cancer Discov; 4(5); 564–77. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 495