Expression of histone deacetylase (HDAC) family members in bortezomib-refractory multiple myeloma and modulation by panobinostat

Aim: Multiple myeloma (MM) is a hematological malignancy of antibody-producing mature B cells or plasma cells. The proteasome inhibitor, bortezomib, was the first-in-class compound to be FDA approved for MM and is frequently utilized in induction therapy. However, bortezomib refractory disease is a major clinical concern, and the efficacy of the pan-histone deacetylase inhibitor (HDACi), panobinostat, in bortezomib refractory disease indicates that HDAC targeting is a viable strategy. Here, we utilized isogenic bortezomib resistant models to profile HDAC expression and define baseline and HDACi-induced expression patterns of individual HDAC family members in sensitive vs. resistant cells to better understanding the potential for targeting these enzymes.Methods: Gene expression of HDAC family members in two sets of isogenic bortezomib sensitive or resistant myeloma cell lines was examined. These cell lines were subsequently treated with HDAC inhibitors: panobinostat or vorinostat, and HDAC expression was evaluated. CRISPR/Cas9 knockdown and pharmacological inhibition of specific HDAC family members were conducted.Results: Interestingly, HDAC6 and HDAC7 were significantly upregulated and downregulated, respectively, in bortezomib-resistant cells. Panobinostat was effective at inducing cell death in these lines and modulated HDAC expression in cell lines and patient samples. Knockdown of HDAC7 inhibited cell growth while pharmacologically inhibiting HDAC6 augmented cell death by panobinostat.Conclusion: Our data revealed heterogeneous expression of individual HDACs in bortezomib sensitive vs. resistant isogenic cell lines and patient samples treated with panobinostat. Cumulatively our findings highlight distinct roles for HDAC6 and HDAC7 in regulating cell death in the context of bortezomib resistance

3D tissue-engineered model of Ewing's sarcoma

Despite longstanding reliance upon monolayer culture for studying cancer cells, and numerous advantages from both a practical and experimental standpoint, a growing body of evidence suggests that more complex three-dimensional (3D) models are necessary to properly mimic many of the critical hallmarks associated with the oncogenesis, maintenance and spread of Ewing's sarcoma (ES), the second most common pediatric bone tumor. And as clinicians increasingly turn to biologically-targeted therapies that exert their effects not only on the tumor cells themselves, but also on the surrounding extracellular matrix, it is especially important that preclinical models evolve in parallel to reliably measure antineoplastic effects and possible mechanisms of de novo and acquired drug resistance. Herein, we highlight a number of innovative methods used to fabricate biomimetic ES tumors, encompassing both the surrounding cellular milieu and the extracellular matrix (ECM), and suggest potential applications to advance our understanding of ES biology, preclinical drug testing, and personalized medicine

Gene expression levels of the top 50 ranked genes.

<p>Gene expression levels of the top 50 ranked genes.</p

Comparison of genes either similarly or differentially expressed.

<p>(A) Entire gene set divided into 4 quantiles based on their level of expression to enable determination percentage of similarly expressed genes and differentially expressed genes. (B) Coding gene set divided into 4 quantiles based on their level of expression to enable determination percentage of similarly expressed genes and differentially expressed genes.</p

Lead candidates identified from a high throughput drug screen utilizing the PDXEx screening platform.

<p>A high throughput small molecule screen was performed utilizing the Bcx087 triple negative PDXEx screening platform and the small molecule Anti-Cancer 386 Compound Library (Selleckchem). The ex vivo tumor tissue array was incubate with the drugs at 37oC for 5 days. The top hits were identified as agents having a superior anticancer effect to that of docetaxel and doxorubicin at 250nM.</p

Tissue characteristics of the ex-vivo tumor model.

<p>(A) Bio-printing cells–Tumor cells tagged with a nanoparticle assembly of iron oxide and iron nanoparticles cross-linked with poly-L-lysine (NanoshuttleTM) were dispensed into an ultralow attachment 96 well tissue culture plate placed on a 96 well magnetic drive (n3D Biosciences, Inc). (B) Morphology (4x magnification) of the ex-vivo tumor tissue prints after 5 days of growth at 37°C revealing uniformly sized structures. (C) Proliferative capacity of the PDXEx tissue following 5 days of culture at 37°C.</p

Comparative drug response profile between IBC PDX Bcx017 mouse model and Bcx017 PDXEx tissue model.

<p>A) Actively growing (~200–350 mm3) implants in mice were grouped and treated with the indicated drugs for 21days. The graph summarizes data from a previously published in vivo study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195932#pone.0195932.ref069" target="_blank">69</a>]. B) Tumor cells tagged with a nanoparticle assembly of iron oxide and iron nanoparticles cross-linked with poly-L-lysine (NanoshuttleTM) were bio-printed into ultra low attachment plates. The PDXEx bio-print was treated with the indicated drugs at the indicated dose range for 5 days.</p

Generation of preclinical PDX-derived ex-vivo model.

<p>(A) A freshly harvested PDX tumor from a mouse was finely chopped to release all its cellular content. The released cells are filtered to separated them form fat and necrotic tissue and tagged with a nanoparticle assembly of iron oxide and iron nanoparticles cross-linked with poly-L-lysine (NanoshuttleTM) by an overnight incubation prior to been placed under a magnetic field. (Bio-AssemblerTM) (n3D Biosciences Inc.). (B) The levitating mass of cells developed into a loose unstructured mass by day 2 of incubation and (C) into a more structured compact mass by day 4 of incubation. (D) Immunohistochemistry analysis of PDX tissue and PDXEx tissue revealed a similar tissue architecture and staining for E-cadherin, Vimentin, Ki67 and pSMAD2.</p

Layout of biological pathways common to all 3 PDX tumors.

<p>The Top 25 out of 1116 pathways are presented.</p