Megan A. Bariteau

Selective Inhibition of HDAC1 and HDAC2 as a Potential Therapeutic Option for B-ALL

Purpose: Histone deacetylase inhibitors (HDACi) have recently emerged as efficacious therapies that target epigenetic mechanisms in hematologic malignancies. One such hematologic malignancy, B-cell acute lymphoblastic leukemia (B-ALL), may be highly dependent on epigenetic regulation for leukemia development and maintenance, and thus sensitive to small-molecule inhibitors that target epigenetic mechanisms. Experimental Design: A panel of B-ALL cell lines was tested for sensitivity to HDACi with varying isoform sensitivity. Isoform-specific shRNAs were used as further validation of HDACs as relevant therapeutic targets in B-ALL. Mouse xenografts of B-cell malignancy–derived cell lines and a pediatric B-ALL were used to demonstrate pharmacologic efficacy. Results: Nonselective HDAC inhibitors were cytotoxic to a panel of B-ALL cell lines as well as to xenografted human leukemia patient samples. Assessment of isoform-specific HDACi indicated that targeting HDAC1-3 with class I HDAC-specific inhibitors was sufficient to inhibit growth of B-ALL cell lines. Furthermore, shRNA-mediated knockdown of HDAC1 or HDAC2 resulted in growth inhibition in these cells. We then assessed a compound that specifically inhibits only HDAC1 and HDAC2. This compound suppressed growth and induced apoptosis in B-ALL cell lines in vitro and in vivo, whereas it was far less effective against other B-cell–derived malignancies. Conclusions: Here, we show that HDAC inhibitors are a potential therapeutic option for B-ALL, and that a more specific inhibitor of HDAC1 and HDAC2 could be therapeutically useful for patients with B-ALL. Clin Cancer Res; 21(10); 2348–58. ©2015 AACR.

Abstract PR12: Biological insights into tumor-bone marrow microenvironment interactions derived from a humanized murine model

Interactions with the hematopoietic niche in the bone marrow (BM) microenvironment are essential for hematopoietic stem cell (HSC) self-renewal. In addition, this niche is considered to serve as a sanctuary site for leukemic stem cells during chemotherapy, and contributes to disease relapse. Although many advances have been made in understanding how the niche regulates HSC self-renewal and confers therapy resistance, most of this knowledge is based on genetically engineered murine models. Given the need for models that more closely resemble the human niche, we developed a humanized model in which a scaffold seeded with human BM stromal cells generates a bone microenvironment. Inoculation of these mice with human CD34+-progenitor cells resulted in homing to the human bone environment and the generation of hematopoietic cells of distinct lineages as well as the engraftment of CD34+ cells themselves. The functionality of these humanized niches was further investigated with primary samples obtained from patients diagnosed with MDS, AML, T-ALL and MM, malignancies of which the tumor cells are highly dependent on the BM microenvironment for survival and growth. In addition, by gene-marking MM and T-ALL cells with luciferase and using bioluminescent imaging, we were able to follow tumor burden over time as well as response to therapy. Importantly, in this model, the response of primary MM cells to established anti-MM agents correlates with clinical responses of the respective patients. Moreover, this model allows us to study bidirectional interactions between MM cells and stromal cells and the resulting impact. By analyzing gene expression in human BM stromal cells (CD73+ CD90+ CD105+) that we cultured from scaffolds containing MM tumors, we identified potential novel markers for osteogenesis in MM (e.g. OGN, OMD and ASPN), as well as adhesion molecules (e.g. ITGA2) and extracellular matrix proteins (e.g. STC1 and TGM2). Hence, our model allows to investigate essential interactions within the human BM microenvironment for the development of normal and malignant hematopoiesis and thus for therapy development. This abstract is also presented as Poster B41. Citation Format: Jessica Sigmans, Willy Noort, Coleman Lindsley, Li Pan, Linda Aalders, Megan Bariteau, Benjamin Ebert, Jon Aster, Uli Steidl, Jan Schuringa, Huipin Yuan, Joost de Bruijn, Reinier Raymakers, Henk Lokhorst, Tuna Mutis, Anton Martens, Constantine Mitsiades, Richard Groen. Biological insights into tumor-bone marrow microenvironment interactions derived from a humanized murine model. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr PR12. doi:10.1158/1538-7445.CHTME14-PR12

Abstract 4713: BET bromodomain degradation as a therapeutic strategy in drug-resistant multiple myeloma

Multiple myeloma (MM) is an incurable malignancy with an unmet need for novel therapeutic modalities. Moreover, acquired or de novo resistance to established or novel therapeutics remains a major challenge in this, and other, neoplasias. BET Bromodomain inhibitors (BBIs), including JQ1, have potent anti-MM activity in vitro and in in vivo, but do not provide curative outcome and do not induce apoptosis in most cell types. We sought to investigate dBET, a class of BBIs that induce degradation of BET Bromodomains (BRDs) through CRBN-mediated ubiquitination and proteasomal degradation, in drug-resistant MM. Additionally, we posited that resistance to dBET treatment would emerge through genetic perturbations and wished to uncover potential mechanisms prior to its clinical utilization. To address this, we compared effects of optimized lead compound, dBET6, with JQ1 on a panel of MM cell lines, including clones resistant to JQ1 or bortezomib and assessed viability using CS-BLI/CTG assay and BRD/c-MYC expression by western blot. Using an open-ended unbiased genome-wide CRISPR (clustered regularly interspaced short palindromic repeats)-associated Cas9 approach, we examined whether we could uncover genes associated with resistance to dBET6. MM1.S cells were transduced with Cas9 and pooled lentiviral particles of the GeCKO library, consisting of 2 pooled sgRNA sub-libraries (∼120,000 sgRNAs; targeting ∼19,000 genes and ∼1800 miRNAs). Using this CRISPR/Cas9-based approach we sought to expedite the isolation of MM cells resistant to dBET6. We treated the pool of cells thrice with dBET (≥IC80), allowing regrowth between treatments and maintaining a coverage of 1000 cells/sgRNA. dBET6-resistant cells were processed to quantify sgRNA enrichment or depletion, using deep sequencing. We observed dBET6 to have significantly greater potency against MM cells than JQ1, or its combination with lenalidomide, and that MM1S.CRBN-/- cells were resistant to dBET6. Resistance to neither JQ1 nor Bortezomib conferred resistance to dBET6. We observed dBET6 to induce rapid ( Citation Format: Geoffrey M. Matthews, Yiguo Hu, Michal Sheffer, Paul J. Hengeveld, Dennis L. Buckley, Megan A. Bariteau, Haley Poarch, James E. Bradner, Constantine S. Mitsiades. BET bromodomain degradation as a therapeutic strategy in drug-resistant multiple myeloma. [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 4713.