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© Zocchi et al. Osteosarcoma is the most common primary malignant neoplasm of bone and typically occurs in children and young adults. As a highly metastatic malignancy, 15- 20% of osteosarcoma patients are diagnosed after the tumor has already metastasized (typically to the lungs), which translates to 5-year survival rates of < 40%. Here, we tested the effect of the cyclin-dependent kinase (CDK) inhibitor flavopiridol (alvocidib) in U2OS, SaOS-2, SJSA-1, and 143B osteosarcoma tumor cells in vitro and in vivo. Our results show that flavopiridol can drastically decrease survival in these osteosarcoma cell lines at nanomolar concentrations and induce mitotic catastrophe in p53-null osteosarcomas. We also performed transcriptome analysis (RNA-seq) of flavopiridoltreated osteosarcoma cells, which revealed significant changes in genes coding for proteins involved in cell-cell and cell-matrix adhesions, including cadherin 3 (CDH3) and 4 (CDH4). These transcriptional changes translated to a striking reduction in the ability of osteosarcoma cells to migrate and invade in vitro. Further, in vivo assessment of the effects of flavopiridol on osteosarcoma metastasis resulted in a significant reduction in the number of lung metastases in mice treated with flavopiridol at concentrations that are physiologically tolerable. This study suggests that flavopiridol, likely in combination with other cytotoxic chemotherapeutic agents, may be a promising drug for the treatment of osteosarcoma.

Class-switch DNA recombination (CSR) and somatic hypermutation (SHM), which require activation-induced cytidine deaminase (AID), and plasma cell differentiation, which requires B lymphocyte–induced maturation protein-1 (Blimp-1), are critical for the generation of class-switched and hypermutated (mature) Ab and autoantibody responses. We show that histone deacetylase inhibitors valproic acid and butyrate dampened AICDA/Aicda (AID) and PRDM1/Prdm1 (Blimp-1) mRNAs by upregulating miR-155, miR-181b, and miR-361 to silence AICDA/Aicda, and miR-23b, miR-30a, and miR-125b to silence PRDM1/Prdm1, in human and mouse B cells. This led to downregulation of AID, Blimp-1, and X-box binding protein 1, thereby inhibiting CSR, SHM, and plasma cell differentiation without altering B cell viability or proliferation. The selectivity of histone deacetylase inhibitor–mediated silencing of AICDA/Aicda and PRDM1/Prdm1 was emphasized by unchanged expression of HoxC4 and Irf4 (important inducers/modulators of AICDA/Aicda), Rev1 and Ung (central elements for CSR/SHM), and Bcl6, Bach2, or Pax5 (repressors of PRDM1/Prdm1 expression), as well as unchanged expression of miR-19a/b, miR-20a, and miR-25, which are not known to regulate AICDA/Aicda or PRDM1/Prdm1. Through these B cell–intrinsic epigenetic mechanisms, valproic acid blunted class-switched and hypermutated T-dependent and T-independent Ab responses in C57BL/6 mice. In addition, it decreased class-switched and hypermutated autoantibodies, ameliorated disease, and extended survival in lupus MRL/Faslpr/lpr mice. Our findings outline epigenetic mechanisms that modulate expression of an enzyme (AID) and transcription factors (Blimp-1 and X-box binding protein 1) that are critical to the B cell differentiation processes that underpin Ab and autoantibody responses. They also provide therapeutic proof-of-principle in autoantibody-mediated autoimmunity.

SummaryClass-switch DNA recombination (CSR) is central to the antibody response, in that it changes the immunoglobulin heavy chain (IgH) constant region, thereby diversifying biological effector functions of antibodies. The activation-induced cytidine deaminase (AID)-centered CSR machinery excises and rejoins DNA between an upstream (donor) and a downstream (acceptor) S region, which precede the respective constant region DNA. AID is stabilized on S regions by 14-3-3 adaptors. These adaptors display a high affinity for 5′-AGCT-3′ repeats, which recur in all S regions. However, how 14-3-3, AID, and the CSR machinery target exclusively the donor and acceptor S regions is poorly understood. Here, we show that histone methyltransferases and acetyltransferases are induced by CD40 or Toll-like receptor signaling and catalyze H3K4me3 and H3K9ac/K14ac histone modifications, which are enriched in S regions but do not specify the S region targets of CSR. By contrast, the combinatorial H3K9acS10ph modification specifically marks the S regions set to recombine and directly recruits 14-3-3 adaptors for AID stabilization there. Inhibition of the enzymatic activity of GCN5 and PCAF histone acetyltransferases reduces H3K9acS10ph in S regions, 14-3-3 and AID stabilization, and CSR. Thus, H3K9acS10ph is a histone code that is “written” specifically in S regions and is “read” by 14-3-3 adaptors to target AID for CSR as an important biological outcome.

Stroke is a major cause of death and disability, with very limited treatment option. Cell-based therapies have emerged as potential treatments for stroke. Indeed, studies have shown that transplantation of neural stem cells (NSCs) exerts functional benefits in stroke models. However, graft survival and integration with the host remain pressing concerns with cell-based treatments. The current study set out to investigate those very issues using a human NSC line, NSI-566RSC, in a rat model of ischemic stroke induced by transient occlusion of the middle cerebral artery. Seven days after stroke surgery, those animals that showed significant motor and neurological impairments were randomly assigned to receive NSI-566RSC intracerebral transplants at two sites within the striatum at three different doses: group A (0 cells/µl), group B (5,000 cells/µl), group C (10,000 cells/µl), and group D (20,000 cells/µl). Weekly behavioral tests, starting at seven days and continued up to 8 weeks after transplantation, revealed dose-dependent recovery from both motor and neurological deficits in transplanted stroke animals. Eight weeks after cell transplantation, immunohistochemical investigations via hematoxylin and eosin staining revealed infarct size was similar across all groups. To identify the cell graft, and estimate volume, immunohistochemistry was performed using two human-specific antibodies: one to detect all human nuclei (HuNu), and another to detect human neuron-specific enolase (hNSE). Surviving cell grafts were confirmed in 10/10 animals of group B, 9/10 group C, and 9/10 in group D. hNSE and HuNu staining revealed similar graft volume estimates in transplanted stroke animals. hNSE-immunoreactive fibers were also present within the corpus callosum, coursing in parallel with host tracts, suggesting a propensity to follow established neuroanatomical features. Despite absence of reduction in infarct volume, NSI-566RSC transplantation produced behavioral improvements possibly via robust engraftment and neuronal differentiation, supporting the use of this NSC line for stroke therapy.