BackgroundInhibitor of Growth (ING) proteins are epigenetic “readers” that recognize trimethylated lysine 4 of histone H3 (H3K4Me3) and target histone acetyl transferase (HAT) and histone deacetylase (HDAC) complexes to chromatin.Methods and Principal FindingsHere we asked whether dysregulating two epigenetic pathways with chemical inhibitors showed synergistic effects on breast cancer cell line killing. We also tested whether ING1 could synergize better with chemotherapeutics that target the same epigenetic mechanism such as the HDAC inhibitor LBH589 (Panobinostat) or a different epigenetic mechanism such as 5-azacytidine (5azaC), which inhibits DNA methyl transferases. Simultaneous treatment of breast cancer cell lines with LBH589 and 5azaC did not show significant synergy in killing cells. However, combination treatment of ING1 with either LBH589 or 5azaC did show synergy. The combination of ING1b with 5azaC, which targets two distinct epigenetic mechanisms, was more effective at lower doses and enhanced apoptosis as determined by Annexin V staining and cleavage of caspase 3 and poly-ADP-ribose polymerase (PARP). ING1b plus 5azaC also acted synergistically to increase γH2AX staining indicating significant levels of DNA damage were induced. Adenoviral delivery of ING1b with 5azaC also inhibited cancer cell growth in a murine xenograft model and led to tumor regression when viral concentration was optimized in vivo.ConclusionsThese data show that targeting distinct epigenetic pathways can be more effective in blocking cancer cell line growth than targeting the same pathway with multiple agents, and that using viral delivery of epigenetic regulators can be more effective in synergizing with a chemical agent than using two chemotherapeutic agents. This study also indicates that the ING1 epigenetic regulator may have additional activities in the cell when expressed at high levels.

Cryopreservation of hematopoietic stem/progenitor cell (HPC) grafts and other cell based products to be infused in the future is a common practice. However, cryopreservation and conditions of storage can influence post thaw recovery of cellular products. HPC graft is composed of heterogeneous blood cells. Various assays detect viability based on different principles and their sensitivity differs. Less than 1 in 100,000 CD34+ cells possess in vivo hematopoietic reconstitution potential, therefore determination of viability and recovery need to be interpreted accordingly. We analyzed total 38 graft aliquots stored in liquid nitrogen (auto 10 yrs = 7, Auto 1 yr = 12, allo 10 yrs = 5, Allo 5 yrs = 5, allo 1 yr = 9). The viable CD34+ cell frequency in allo HPC grafts was higher than auto grafts cryopreserved 10 years ago (Auto 10 yrs = 0.39 ± 0.24%, Allo 10 yrs = 0.96 ±0.31%, Allo 5 yrs = 0.82 ± 0.3%, p = 0.018). When we compared both single and dual ISHAGE platform methods it displayed comparable post thaw viable CD34+ cell numbers (Figure 1A). The recovery of viable total nucleated cells (TNC) based on trypan blue dye exclusion was 46.83± 4.98% auto 10 years, 43.11 ± 7.76% allo 10 years, 48.74 ± 4.94% allo 5 years, 87.56 ± 6.93% allo 1 year. The viable CD34+ cell recovery between the groups were comparable (Figure 1B). Auto HPC grafts stored for more than 10 years displayed 64.05±16% while auto grafts stored less than a year yielded 85.4±15.15% viable CD34 recovery. The effects of storage duration on grafts is presented below by monitoring hematopoietic engraftment after transplantation. Interestingly, auto grafts in comparison to allo grafts stored for 5 to10 years displayed higher red fraction (JC-1); indicative of higher mitochondrial membrane potential (MMP) and viability (Auto 10 yrs = 72.24 ± 12.45%, Allo 10 yrs = 40.6 ± 13.92%, Allo 5 ysr = 47.2 ± 15.36%, p = 0.011). The higher fraction of cells with intact MMP (red fraction) within CD34+ cell compartment of post thaw suggesting likely higher resilience of auto grafts. Samples stored up to10 years displayed positive colony forming unit (CFU) growth but had poor CFU recovery. CFU recovery for HPC grafts stored for less than 1 year displayed 82.93 ± 25.68% for auto grafts while allo graft displayed 72.46 ± 34.76%. HPC grafts stored for 5 to 10 years which were utilized here to determine graft stability were transplanted within two months of storage and displayed timely neutrophil and platelet engraftment. When single ISHAGE platform method was used to enumerate viable CD34+ cells and HPC grafts are thawed within one year, viable CD34 recovery for auto HPC graft was 85.4 ± 15.15% and for allo graft recovery was 86.85 ±18.0%. Four patients which were transplanted (CD34+ cells infused mean ± SD, 4.35 ± 1.43 x 10e6/KG body weight) with autologous grafts stored for 5 to 8 years in liquid nitrogen (vapor phase) displayed timely neutrophil (9.5 ± 1 day) and platelet (16.5 ± 5 day) engraftment. Taken together, viability assays detect various aspects of cell integrity. The post thaw viable CD34 recovery corresponds well with in vivo hematopoietic reconstitution but in some instances functional potency assays particularly for samples stored longer than 5 years may be crucial. The relatively higher resilience of auto HPC grafts stored for longer than10 years is of note which will need to be further validated. Disclosures Patel: Celgene: Speakers Bureau; Janssen: Speakers Bureau; Amgen: Consultancy, Speakers Bureau.

Eukaryotic initiation factor subunit I (EIF3i), also called as p36 or TRIP-1, is a component of the translation initiation complex and acts as a modulator of TGF-β signaling. We demonstrated earlier that this intracellular protein is not only exported to the extracellular matrix via exosomes but also binds calcium phosphate and promotes hydroxyapatite nucleation. To assess other functional roles of TRIP-1, we first examined their phylogeny and showed that it is highly conserved in eukaryotes. Comparing human EIF3i sequence with that of 63 other eukaryotic species showed that more than 50% of its sequence is conserved, suggesting the preservation of its important functional role (translation initiation) during evolution. TRIP-1 contains WD40 domains and predicting its function based on this structural motif is difficult as it is present in a vast array of proteins with a wide variety of functions. Therefore, bioinformatics analysis was performed to identify putative regulatory functions for TRIP-1 by examining the structural domains and post-translational modifications and establishing an interactive network using known interacting partners such as type I collagen. Insight into the function of TRIP-1 was also determined by examining structurally similar proteins such as Wdr5 and GPSß, which contain a ß-propeller structure which has been implicated in the calcification process. Further, proteomic analysis of matrix vesicles isolated from TRIP-1-overexpressing preosteoblastic MC3T3-E1 cells demonstrated the expression of several key biomineralization-related proteins, thereby confirming its role in the calcification process. Finally, we demonstrated that the proteomic signature in TRIP1-OE MVs facilitated osteogenic differentiation of stem cells. Overall, we demonstrated by bioinformatics that TRIP-1 has a unique structure and proteomic analysis suggested that the unique osteogenic cargo within the matrix vesicles facilitates matrix mineralization.

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The treatment outcomes for patients diagnosed with acute myeloid leukemia (AML) are still dismal. Recent advances in understanding AML indicate that the lack of efficacy is primarily due to non-specificity of currently used chemotherapeutics targeting both leukemic stem/progenitor cells (LSC) and normal hematopoietic stem cells (HSC). Thus, a critical barrier is the identification of innovative therapies that selectively target LSC. Histone deacetylase 8 (HDAC8) has been shown to enhance p53 protein deacetylation, which results in inactivation of p53, promoting LSC survival. We hypothesize that enzymatic/non-enzymatic role of HDAC8 is critical for LSC survival but not for HSCs. Then, we characterized our two tetrahydroisoquinoline (TIQ)-based selective HDAC8 inhibitors (HDAC8i) BIP and OCH3 for growth inhibition, apoptosis, activation of caspase 3, integrity of mitochondrial membrane potential (MMP), and acetylation of histone H4 in human leukemia cell lines. The growth inhibitory effects observed in cell lines were validated using bone marrow (BM) or peripheral blood (PB) cells from AML patients. Colony forming cell (CFC) assays were performed using AML BM/PB cells treated with OCH3 or BIP. OCH3 and BIP were also tested for hematotoxicity using normal CB CD34+ cells. Furthermore, we compared class I HDAC isoform engagement in human normal cord blood (CB) CD34+ cells and in SET-2 leukemia cells using our novel photoreactive probe TH1143. In CD34+ cells, TH1143 had higher level of engagement for HDAC1 and 2, whereas engagement of HDAC3 and 8 was minimal. In SET-2 cells, HDAC3 and HDAC8 displayed relatively higher engagement with TH1143 indicating HDAC engagement is likely cell type specific. The biological efficacies of OCH3 at 50uM and BIP at 25uM were noted to exert >50% growth inhibition in KG1 and in K562 leukemia cells. Both OCH3 and BIP significantly increased the number of apoptotic cells and there was an enhanced active caspase-3 activity. Furthermore, OCH3 and BIP treated cells displayed lower red/green ratio in comparison to control, indicative of poor MMP and depolarization to induce apoptosis (Table 1.a). OCH3 and BIP were further validated by using BM/PB cells from AML patients showing growth inhibition. This was also accompanied by increase in apoptotic cells by OCH3 and BIP. In contrast to BIP, OCH3 spared CB CD34+ cells as demonstrated by notably lower growth inhibition, apoptotic cells vs control when compared with primary AML cells from patients. Both OCH3 and BIP displayed minimal inhibition of CFU growth in CD34+ cells. However, HDAC8i induced significant CFU growth inhibition in primary AML samples suggesting that HDAC8i spares normal CFU progenitors but not leukemia progenitors (Table 1.b). Notably, both BIP and OCH3 lack ability to exert acetylation of histone H4, unlike broad spectrum HDAC inhibitor TSA (MFI with OCH3=0.96±0.03, BIP=0.77±0, TSA =1.63±0.15) which is consistent with isoform selectivity of OCH3 and BIP. The leukemia growth inhibitory effects at LSC level was demonstrated using ex vivo OCH3 treated AML patient derived BM/PB cells transplantation in humanized immunodeficient NSGS mice. After 10 to 12 weeks of transplantation mice receiving untreated AML cells had 7.73±2.18% while with OCH3 treatment mice had 4.84±1.37% human CD34+ leukemia cells, a 38% reduction in CD34+ leukemia cells, despite only a single ex vivo exposure to OCH3. Furthermore, in a second model, NSGS humanized mice were transplanted (IV) with primary leukemia cells from AML patients and after 4 weeks injected (IP) with OCH3 or vehicle control. After 12 weeks of transplantation in this second model human primary AML cell burden was 5.74±1.31% (OCH3) and 18.13±12.76% (vehicle control), while mice transplanted with normal CD34+ cells treated similarly with OCH3 or vehicle control displayed no detectable inhibition of human myeloid cell chimerism (OCH3:12.28 ± 3.31% vs vehicle control: 17.92±11.96%). Taken together, our data indicate that HDAC8 isoform inhibitor, OCH3 displayed significant inhibition of primary AML patient derived leukemia cells growth in vitro and in vivo in contrast to normal CD34+ cells. Selective inhibition of HDAC8 is sufficient to cause growth inhibition in primary AML progenitors including LSCs in vivo while sparing normal HSCs thus offer opportunities for further development of HDAC8i as new experimental therapeutics in AML. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

No abstract

Dentin phosphophoryn synthesized and processed predominantly by the odontoblasts, functions as both structural and signaling protein. Mechanistic studies revealed that DPP stimulation of DPSCs positively impacted the differentiation of DPSCs into functional odontoblasts. Results show that NF-κB signaling and transcriptional activation of genes involved in odontoblast differentiation were influenced by DPP signaling. Specifically, RelA/p65 subunit of NF-κB was identified as being responsible for the initiation of the differentiation cascade. Confocal imaging demonstrated the nuclear translocation of p65 with DPP stimulation. Moreover, direct binding of nuclear NF-κB p65 subunit to the promoter elements of Runx2, Osx, OCN, MMP1, MMP3, BMP4 and PTX3 were identified by ChIP analysis. Pharmacological inhibition of the NF-κB pathway using TPCA-1, a selective inhibitor of IKK-2 and JSH-23, an inhibitor that prevents nuclear translocation and DNA binding of p65 showed impairment in the differentiation process. Functional studies using Alizarin-Red staining showed robust mineral deposits with DPP stimulation and sparse deposition with defective odontoblast differentiation in the presence of inhibitors. In vivo expression of NF-κB targets such as OSX, OCN, PTX3 and p65 in odontoblasts and dental pulp cells from DSPP null mouse was lower when compared with the wild-type. Overall, the results suggest an important role for DPP-mediated NF-κB activation in the transcriptional regulation of early odontogenic markers that promote differentiation of DPSCs.

The limited number of hematopoietic stem cells (HSC) within a single unit of human cord blood currently limits its use as an alternate graft source. However, we have developed a strategy using 5-aza-29-deoxycytidine (5azaD) and trichostatin A (TSA), which expands transplantable HSC 7-to 10-fold. In our current studies, we have assessed the allostimulatory capacity of the 5azaD/TSA-expanded grafts. The coexpression of immunophenotypic dendritic cell (DC) markers, such as HLA-DR/CD86 and HLA-DR/CD11c as determined by flow cytometry, and the allostimulatory capacity of 5azaD/TSA-expanded cells as determined by MLC were both significantly lower than control. It has been previously demonstrated that STAT3 is indispensable for the differentiation of DC from HSC. Real-time quantitative PCR analysis revealed that 5azaD/TSA-expanded cells expressed more STAT3 transcript than control while also expressing increased transcripts for STAT3 inhibitors including SHP1, p21, and GATA1. Western blot analysis indicates that chromatin-modifying agent-expanded grafts displayed a reduced ratio of p-STAT3 to total STAT3 than control cultures, which is likely indicative of STAT3 inactivity in 5azD/TSA-expanded grafts. Culturing 5azaD/ TSA-expanded cord blood cells in extended cultures reveals that they are still capable of generating DC. Notably, STAT3 inactivity was transient because the transcript levels of STAT3 and its inhibitors, including SHP1, were comparable between 5azaD/TSA and control cultures following extended culture. Taken together, our studies indicate that the reduced allostimulatory capacity of 5azaD/TSA-expanded cells is likely because of reversible inhibition of STAT3-dependent DC differentiation. These results suggest that a graft composed of 5azaD/TSA-expanded cells possesses relatively less allostimulatory response but is still capable of generating DC in permissive conditions.