Tamar Uziel

The miR-17∼92 cluster collaborates with the Sonic Hedgehog pathway in medulloblastoma

Medulloblastomas (MBs) are the most common brain tumors in children. Some are thought to originate from cerebellar granule neuron progenitors (GNPs) that fail to undergo normal cell cycle exit and differentiation. Because microRNAs regulate numerous aspects of cellular physiology and development, we reasoned that alterations in miRNA expression might contribute to MB. We tested this hypothesis using 2 spontaneous mouse MB models with specific initiating mutations, Ink4c−/−; Ptch1+/− and Ink4c−/−; p53−/−. We found that 26 miRNAs showed increased expression and 24 miRNAs showed decreased expression in proliferating mouse GNPs and MBs relative to mature mouse cerebellum, regardless of genotype. Among the 26 overexpressed miRNAs, 9 were encoded by the miR-17∼92 cluster family, a group of microRNAs implicated as oncogenes in several tumor types. Analysis of human MBs demonstrated that 3 miR-17∼92 cluster miRNAs (miR-92, miR-19a, and miR-20) were also overexpressed in human MBs with a constitutively activated Sonic Hedgehog (SHH) signaling pathway, but not in other forms of the disease. To test whether the miR-17∼92 cluster could promote MB formation, we enforced expression of these miRNAs in GNPs isolated from cerebella of postnatal (P) day P6 Ink4c−/−; Ptch1+/− mice. These, but not similarly engineered cells from Ink4c−/−; p53−/− mice, formed MBs in orthotopic transplants with complete penetrance. Interestingly, orthotopic mouse tumors ectopically expressing miR-17∼92 lost expression of the wild-type Ptch1 allele. Our findings suggest a functional collaboration between the miR-17∼92 cluster and the SHH signaling pathway in the development of MBs in mouse and man.

A mouse model of the most aggressive subgroup of human medulloblastoma

Medulloblastomas that display a large cell/anaplastic morphology and overexpress the cellular c-MYC gene are highly aggressive and carry a very poor prognosis. This so-called MYC-subgroup differs in its histopathology, gene expression profile, and clinical behavior from other forms of medulloblastoma. We generated a mouse model of MYC-subgroup medulloblastoma by transducing Trp53-null cerebellar progenitor cells with Myc. The cardinal features of these mouse medulloblastomas closely mimic those of human MYC-subgroup tumors and significantly differ from mouse models of the Sonic-Hedgehog- and WNT-disease subgroups. This mouse model should significantly accelerate understanding and treatment of the most aggressive form of medulloblastoma and infers distinct roles for MYC and MYCN in tumorigenesis.

The CDK inhibitor p18Ink4c is a tumor suppressor in medulloblastoma.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor which is thought to originate from cerebellar granule cell precursors (CGNPs) that fail to properly exit the cell cycle and differentiate. Although mutations in the Sonic Hedgehog (Shh) signaling pathway occur in ~30% of cases, genetic alterations that account for MB formation in most patients have not yet been identified. We recently determined that the cyclin D-dependent kinase inhibitor, p18Ink4c, is expressed as CGNPs exit the cell cycle, suggesting that this protein might play a central role in arresting the proliferation of these cells and in timing their subsequent migration and differentiation. In mice, disruption of Ink4c collaborates independently with loss of p53 or with inactivation of the gene (Ptc1) encoding the Shh receptor, Patched, to induce MB formation. Whereas loss of both Ink4c alleles is required for MB formation in a p53-null background, Ink4c is haplo-insufficient for tumor suppression in a Ptc1+/- background. Moreover, MBs derived from Ptc1+/- mice that lack one or two Ink4c alleles retain wild-type p53. Methylation of the INK4C (CDKN2C) promoter and complete loss of p18INK4C protein expression were detected in a significant fraction of human MBs again pointing toward a role for INK4C in suppression of MB formation.

Exploitation of Castration-Resistant Prostate Cancer Transcription Factor Dependencies by the Novel BET Inhibitor ABBV-075

Competitive inhibitors of acetyl-lysine binding to the bromodomains of the BET (bromodomain and extra terminal) family are being developed for the treatment of solid and hematologic malignancies. The function of BET family member BRD4 at enhancers/superenhancers has been shown to sustain signal-dependent or pathogenic gene expression programs. Here, the hypothesis was tested that the transcription factor drivers of castration-resistant prostate cancer (CRPC) clinical progression, including the androgen receptor (AR), are critically dependent on BRD4 and thus represent a sensitive solid tumor indication for the BET inhibitor ABBV-075. DHT-stimulated transcription of AR target genes was inhibited by ABBV-075 without significant effect on AR protein expression. Furthermore, ABBV-075 disrupted DHT-stimulated recruitment of BET family member BRD4 to gene-regulatory regions cooccupied by AR, including the well-established PSA and TMPRSS2 enhancers. Persistent BET inhibition disrupted the composition and function of AR-occupied enhancers as measured by a reduction in AR and H3K27Ac ChIP signal and inhibition of enhancer RNA transcription. ABBV-075 displayed potent antiproliferative activity in multiple models of resistance to second-generation antiandrogens and inhibited the activity of the AR splice variant AR-V7 and ligand-binding domain gain-of-function mutations, F877L and L702H. ABBV-075 was also a potent inhibitor of MYC and the TMPRSS2-ETS fusion protein, important parallel transcription factor drivers of CRPC. Implications: The ability of BET family inhibitor ABBV-075 to inhibit transcription activation downstream of the initiating events of transcription factors like AR and TMPRSS2:ETS fusion proteins provides a promising therapeutic option for CRPC patients who have developed resistance to second-generation antiandrogens. Mol Cancer Res; 15(1); 35–44. ©2016 AACR.

HEXIM1 as a robust pharmacodynamic marker for monitoring target engagement of BET family bromodomain inhibitors in tumors and surrogate tissues

An increasing number of BET family protein inhibitors have recently entered clinical trials. It has been reported that attempts of monitoring target engagement of the BET bromodomain inhibitor OTX015 using literature-described putative pharmacodynamic markers, such as c-Myc, BRD2, etc., failed to detect pharmacodynamic marker responses in AML patients treated at active dose and those with clinical responses. Here, we report the identification and characterization of HEXIM1 and other genes as robust pharmacodynamic markers for BET inhibitors. Global gene expression profiling studies were carried out using cancer cells and surrogate tissues, such as whole blood and skin, to identify genes that are modulated by BET family proteins. Candidate markers were further characterized for concentration- and time-dependent responses to the BET inhibitor ABBV-075 in vitro and in vivo. HEXIM1 was found to be the only gene that exhibited robust and consistent modulation by BET inhibitors across multiple cancer indications and surrogate tissues. Markers such as SERPINI1, ZCCHC24, and ZMYND8 were modulated by ABBV-075 and other BET inhibitors across cancer cell lines and xenograft tumors but not in blood and skin. Significant downregulation of c-Myc, a well-publicized target of BET inhibitors, was largely restricted to hematologic cancer cell lines. Incorporating well-characterized pharmacodynamic markers, such as HEXIM1 and other genes described here, can provide a better understanding of potential efficacy and toxicity associated with inhibiting BET family proteins and informs early clinical decisions on BET inhibitor development programs. Mol Cancer Ther; 16(2); 388–96. ©2016 AACR.

Preclinical Characterization of BET Family Bromodomain Inhibitor ABBV-075 Suggests Combination Therapeutic Strategies

ABBV-075 is a potent and selective BET family bromodomain inhibitor that recently entered phase I clinical trials. Comprehensive preclinical characterization of ABBV-075 demonstrated broad activity across cell lines and tumor models, representing a variety of hematologic malignancies and solid tumor indications. In most cancer cell lines derived from solid tumors, ABBV-075 triggers prominent G1 cell-cycle arrest without extensive apoptosis. In this study, we show that ABBV-075 efficiently triggers apoptosis in acute myeloid leukemia (AML), non-Hodgkin lymphoma, and multiple myeloma cells. Apoptosis induced by ABBV-075 was mediated in part by modulation of the intrinsic apoptotic pathway, exhibiting synergy with the BCL-2 inhibitor venetoclax in preclinical models of AML. In germinal center diffuse large B-cell lymphoma, BCL-2 levels or venetoclax sensitivity predicted the apoptotic response to ABBV-075 treatment. In vivo combination studies uncovered surprising benefits of low doses of ABBV-075 coupled with bortezomib and azacitidine treatment, despite the lack of in vitro synergy between ABBV-075 and these agents. The in vitro/in vivo activities of ABBV-075 described here may serve as a useful reference to guide the development of ABBV-075 and other BET family inhibitors for cancer therapy. Cancer Res; 77(11); 2976-89. ©2017 AACR.

Vulnerability of small cell lung cancer to apoptosis induced by the combination of BET bromodomain proteins and BCL2 inhibitors

Ten percent to 15% of all lung cancers are small-cell lung cancer (SCLC). SCLC usually grows and metastasizes before it is diagnosed and relapses rapidly upon treatment. Unfortunately, no new targeted agent has been approved in the past 30 years for patients with SCLC. The BET (bromodomain and extraterminal) proteins bind acetylated histones and recruit protein complexes to promote transcription initiation and elongation. BET proteins have been shown to regulate expression of key genes in oncogenesis, such as MYC, CCND2, and BCL2L1. Here, we demonstrate that approximately 50% of SCLC cell lines are exquisitely sensitive to growth inhibition by the BET inhibitor, ABBV-075. The majority of these SCLC cell lines underwent apoptosis in response to ABBV-075 treatment via induction of caspase-3/7 activity. ABBV-075 enhanced the expression of proapoptotic protein BIM and downregulated antiapoptotic proteins BCL2 and BCLxl to a lesser extent. Furthermore, BET inhibition increased BCL2–BIM complex, thus priming the cells for apoptosis. Indeed, strong synergy was observed both in vitro and in vivo when cotreating the cells with BET inhibitor and the BH3-mimetic, BCL2 inhibitor venetoclax (ABT-199). ABBV-075 interaction with venetoclax positively correlated with BCL2 expression. Taken together, our studies provide a rationale for treating SCLC with BET and BCL2 inhibitors in tumors with high BCL2 protein expression. Mol Cancer Ther; 16(8); 1511–20. ©2017 AACR.

Abstract 4718: ABBV-075, a novel BET family bromodomain inhibitor, represents a promising therapeutic agent for a broad spectrum of cancer indications

Small molecule inhibitors of the bromodomain and extraterminal domain (BET) proteins have emerged as a promising option for cancer therapy. ABBV-075 is a potent and selective BET family bromodomain inhibitor that recently entered Phase 1 clinical trials. It binds bromodomains of BRD2/4/T with similar affinities (Ki of 1-2.2 nM), but exhibits roughly 10-fold weaker potency towards BRD3 (Ki of 12.2 nM). ABBV-075 is highly selective for 18 bromodomain proteins tested (Kd > 1 μM; more than 600-fold selectivity vs. BRD4) and has moderate activity towards CREBBP (Kd = 87 μM; 54-fold selectivity vs. BRD4). ABBV-075 exhibited robust single agent activity in cell viability assays across cancer cell lines derived from solid tumors, leukemia and lymphomas. Further characterization of cancer cell responses to ABBV-075 indicated that ABBV-075 manifested diverse mechanisms of action in different cancer settings. These include 1): disruption of cell cycle control leading to G1 arrest followed by senescence, 2) inhibition of oncogenesis drivers leading to apoptosis and 3) potentially targeting tumor microenvironment to provide additional therapeutic benefit. Consistent with its broad spectrum of activities in vitro, ABBV-075 has comparable or superior efficacies to standard of care agents in flank xenograft mouse models of non-small-cell and small cell lung cancers, pancreatic, breast, prostate, head & neck cancers, multiple myeloma, diffuse large B cell lymphoma and leukemia. These results support the development of ABBV-075 in diverse hematological malignancies and solid tumor indications. Citation Format: Aparna Sarthy, Leiming Li, Daniel H. Albert, Xiaoyu Lin, Warder Scott, Emily Faivre, Mai H. Bui, Xiaoli Huang, Denise M. Wilcox, Terry Magoc, Fritz G. Buchanan, Paul Tapang, George S. Sheppard, Le Wang, Steve D. Fidanze, John Pratt, Dachun Liu, Lisa Hasvold, Paul Hessler, Tamar Uziel, Lloyd Lam, Ganesh Rajaraman, Guowei Fang, Steven W. Elmore, Saul H. Rosenberg, Keith McDaniel, Warren Kati, Yu Shen. ABBV-075, a novel BET family bromodomain inhibitor, represents a promising therapeutic agent for a broad spectrum of cancer indications. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4718.

LRRC15 is a novel mesenchymal protein and stromal target for antibody-drug conjugates.

Progress in understanding tumor stromal biology has been constrained in part because cancer-associated fibroblasts (CAF) are a heterogeneous population with limited cell-type-specific protein markers. Using RNA expression profiling, we identified the membrane protein leucine-rich repeat containing 15 (LRRC15) as highly expressed in multiple solid tumor indications with limited normal tissue expression. LRRC15 was expressed on stromal fibroblasts in many solid tumors (e.g., breast, head and neck, lung, pancreatic) as well as directly on a subset of cancer cells of mesenchymal origin (e.g., sarcoma, melanoma, glioblastoma). LRRC15 expression was induced by TGFβ on activated fibroblasts (αSMA+) and on mesenchymal stem cells. These collective findings suggested LRRC15 as a novel CAF and mesenchymal marker with utility as a therapeutic target for the treatment of cancers with LRRC15-positive stromal desmoplasia or cancers of mesenchymal origin. ABBV-085 is a monomethyl auristatin E (MMAE)-containing antibody-drug conjugate (ADC) directed against LRRC15, and it demonstrated robust preclinical efficacy against LRRC15 stromal-positive/cancer-negative, and LRRC15 cancer-positive models as a monotherapy, or in combination with standard-of-care therapies. ABBV-085s unique mechanism of action relied upon the cell-permeable properties of MMAE to preferentially kill cancer cells over LRRC15-positive CAF while also increasing immune infiltrate (e.g., F4/80+ macrophages) in the tumor microenvironment. In summary, these findings validate LRRC15 as a novel therapeutic target in multiple solid tumor indications and support the ongoing clinical development of the LRRC15-targeted ADC ABBV-085.Significance: These findings identify LRRC15 as a new marker of cancer-associated fibroblasts and cancers of mesenchymal origin and provide preclinical evidence for the efficacy of an antibody-drug conjugate targeting the tumor stroma. Cancer Res; 78(14); 4059-72. ©2018 AACR.

Abstract 800: ABBV-744, a first-in-class and highly selective inhibitor of the second bromodomain of BET family proteins, displays robust activities in preclinical models of acute myelogenous leukemia

Many small-molecule inhibitors that target both bromodomains of the BET family proteins (pan BET inhibitors) are undergoing studies in clinical trials. Emerging data are beginning to suggest that clinical responses to these pan BET inhibitors in subsets of hematologic malignancies may be modest and short lived, perhaps due, at least in part, to tolerability issues that limit dosing levels. We hypothesized that selective inhibition of four of the eight bromodomains in BET family proteins might retain the anticancer activities in certain tumor subsets while alleviating some of the tolerability liabilities of pan BET inhibitors, thus possibly providing better therapeutic benefits. ABBV-744 is a highly selective inhibitor for the second bromodomain (BDII) of the four BET family proteins, exhibiting greater than 300-fold more potent binding affinity to the BDII bromodomain of BRD4 relative to the first bromodomain (BDI) of BRD4. In contrast to the broad antiproliferative activities observed with pan BET inhibitors, ABBV-744 only displayed significant antiproliferative activities in a limited number of cancer cell lines, including AML and androgen receptor (AR)-positive prostate cancer. Studies in AML xenograft models demonstrated antitumor efficacy for ABBV-744 that was comparable to the pan-BET inhibitor ABBV-075 but with improved tolerability. Taken together, these results suggest that ABBV-744 could be a promising second-generation BET inhibitor for AML therapy. Affiliation: Oncology Discovery, AbbVie Inc., 1 North Waukegan Rd., North Chicago, IL 60064. Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication. Citation Format: Xiaoyu Lin, Xiaoli Huang, Richard Bellin, Emily Faivre, Paul Hessler, Lloyd Lam, Mai Ha Bui, Denise Wilcox, Tamar Uziel, Debra C. Ferguson, Terrance J. Magoc, Daniel H. Albert, Keith F. McDaniel, Warren Kati, Yu Shen. ABBV-744, a first-in-class and highly selective inhibitor of the second bromodomain of BET family proteins, displays robust activities in preclinical models of acute myelogenous leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 800.