Chromatin-modifying Agents Research Articles

The neural plasticity of early-passage human bone marrow-derived mesenchymal stem cells and their modulation with chromatin-modifying agents

Mesenchymal stem cells (MSCs) in their immature state express a variety of genes of the three germ layers at relatively low or moderate levels that might explain their phenomenal plasticity. Numerous recent studies have demonstrated that under the appropriate conditions in vitro and in vivo the expression of different sets of these genes can be upregulated, turning MSCs into variety of cell lineages of mesodermal, ectodermal and endodermal origin. While transdifferentiation of MSCs is still controversial, these unique properties make MSCs an ideal autologous source of easily reprogrammable cells. Recently, using the approach of cell reprogramming by biological active compounds that interfere with chromatin structure and function, as well as with specific signalling pathways that promote neural fate commitment, we have been able to generate neural-like cells from human bone marrow (BM)-derived MSCs (hMSCs). However, the efficiency of neural transformation of hMSCs induced by this approach gradually declined with passaging. To elucidate the mechanisms that underlie the higher plasticity of early-passage hMSCs, comparative analysis of the expression levels of several pluripotent and neural genes was conducted for early- and late-passage hMSCs. The results demonstrated that early-passage hMSCs expressed the majority of these genes at low and moderate levels that gradually declined at late passages. Neural induction further increased the expression of some of these genes in hMSCs, accompanied by morphological changes into neural-like cells. We concluded that low and moderate expression of several pluripotent and neural genes in early-passage hMSCs could explain their higher plasticity and pliability for neural induction.

Chromatin-modifying agents in anti-cancer therapy

Epigenetic alterations are involved in every step of carcinogenesis. The development of chromatin-modifying agents (CMAs) has provided the ability to fight cancer by reversing these alterations. Currently, four CMAs have been approved for cancer treatment; two DNA demethylating agents and two deacetylase inhibitors. A number of promising CMAs are undergoing clinical trials in several cancer types. Moreover, already approved CMAs are still under clinical investigation to improve their efficacy and to extend their use to a broader panel of cancers. Combinatorial treatments with CMAs are already considered a promising strategy to improve clinical benefits and to limit side effects. The real mechanisms by which these CMAs allow the improvement and remission of patients are still obscure. A deeper analysis of the molecular features expressed by responding patients should be performed to reveal this information. In this review, we focus on clinical trials with CMAs, discussing the success and the pitfalls of this new class of anti-cancer drugs.

Treatment of Nuclear-Donor Cells or Cloned Zygotes with Chromatin-Modifying Agents Increases Histone Acetylation But Does Not Improve Full-Term Development of Cloned Cattle

Although somatic cell nuclear transfer (SCNT) is a promising tool, its potential use is hampered by the high mortality rates during the development to term of cloned offspring. Abnormal epigenetic reprogramming of donor nuclei after SCNT is thought to be the main cause of this low efficiency. We hypothesized that chromatin-modifying agents (CMAs) targeting chromatin acetylation and DNA methylation could alter the chromatin configuration and turn them more amenable to reprogramming. Thus, bovine fibroblasts were treated with 5-aza-2'-deoxycytidine (AZA) plus trichostatin (TSA) or hydralazine (HH) plus valproic acid (VPA) whereas, in another trial, cloned bovine zygotes were treated with TSA. The treatment of fibroblasts with either AZA+TSA or HH+VPA increased histone acetylation, but did not affect the level of DNA methylation. However, treatment with HH+VPA decreased cellular viability and proliferation. The use of these cells as nuclear donors showed no positive effect on pre- and postimplantation development. Regarding the treatment of cloned zygotes with TSA, treated one-cell embryos showed an increase in the acetylation patterns, but not in the level of DNA methylation. Moreover, this treatment revealed no positive effect on pre- and postimplantation development. This work provides evidence the treatment of either nuclear donor cells or cloned zygotes with CMAs has no positive effect on pre- and postimplantation development of cloned cattle.

Ex Vivo Expansion of Human Mobilized Peripheral Blood Stem Cells Using Epigenetic Modifiers

Abstract 1920Currently, a significant percentage of hematopoietic stem cell (HSC) transplantations are being performed using growth factor mobilized peripheral blood (MPB) grafts. Unfortunately, about 5 to 40% of patients are unable to benefit from HSC transplantation due to failure to mobilize and harvest an adequate graft (> 2 × 106 CD34+ cells/kg). Epigenetic modifications are thought to be important in determining the fate of HSC including self renewal and differentiation. We have previously demonstrated that sequential addition of chromatin modifying agents (CMA), 5-aza-2’-deoxyctidine (5azaD) and trichostatin A (TSA), is capable of expanding transplantable HSC 7-fold from human cord blood (CB), likely by preventing the silencing of genes which promote HSC self renewal divisions (Araki et al. Blood 2007). Using the same protocol we have also previously shown that 5azaD/TSA can expand CD34+CD90+ cells containing in vivo repopulating capacity from human bone marrow (BM) 2.5-fold (Milhem et al. Blood 2004). The objectives of our current studies were to assess whether CMA can also expand HSCs present in MPB. In order to test this hypothesis, CD34+ cells were isolated from MPB products from three healthy donors and were expanded ex vivo using 5azaD/TSA for 9 days as described previously (Araki et al. Blood 2007). Following culture, expansion of primitive CD34+CD90+ cells, colony forming unit mixed lineages (CFU-mix), and long term (5 weeks) cobblestone area forming cells (CAFC) were assessed. A 3.74 ± 0.77 fold expansion of CD34+CD90+ cells was observed in 5azaD/TSA expanded MPB cells while only a 0.93 ± 0.23 fold expansion was observed in control cultures (p = 0.025). The 5azaD/TSA expanded MPB cells had a 10.1-fold increase in the number of CFU-mix in comparison to no expansion in the control cultures (p = 0.0055). A 2.26-fold expansion of CAFC numbers was observed in 5azaD/TSA expanded MPB cells in comparison to 0.19-fold expansion in control cultures. Taken together, our data indicate that 5azaD/TSA can expand MPB CD34+CD90+ cells 3.74-fold which also possess the functional capacity to generate primitive CFU-mix and long term CAFCs. This expansion of primitive MPB CD34+CD90+ cells appears to be at an intermediate level (3.74 fold) in comparison to BM and CB which had 2.5-fold and 10.5-fold expansion, respectively. We have previously demonstrated that CD34+CD90+ expanded CB cells are exclusively responsible for reconstituting blood cells following transplantation (Araki et al. Exp Hematol 2006). Currently, the frequency of in vivo repopulating units for CMA expanded MPB is being determined in contrast to expanded BM and CB cells. However, it remains to be investigated what determines the limit for ex vivo expansion of HSC by epigenetic modifiers based on their ontogeny. Towards this goal we analyzed transcription levels of several genes implicated for HSC self renewal/expansion including HoxB4, GATA 2, and Ezh2, which were compared between MPB cells prior to and following expansion in 5azaD/TSA or control cultures. Significantly higher transcript levels were detected for HoxB4 (p = 0.003), GATA 2 (p = 0.0002), and Ezh2 (p = 0.0001) by real time quantitative RT PCR in the 5azaD/TSA expanded MPB graft in comparison to control cultures. Interestingly the transcript levels of HoxB4 and GATA 2 but not Ezh2 were significantly lower in expanded cells in contrast to unmanipulated primary MPB cells. This is in sharp contrast to our earlier results from CB in which 5azaD/TSA expanded cells displayed much higher transcript levels of HoxB4 and GATA 2 compared to primary unmanipulated CB cells. Previously we have demonstrated that environmental conditions can influence the degree of expansion of transplantable HSC from CB (Araki et al. Exp Hematol 2009). Using the same protocol we expanded MPB cells in the presence or absence of CMA using either optimal (SCF, TPO, FLT3L) or suboptimal cytokine cocktails (SCF, TPO, FLT3L with IL-3 and IL-6). Interestingly, unlike CB cells no significant difference in expansion between the two cytokine groups with or without CMA was observed (4.5 versus 4.3-fold expansion of CD34+CD90+ cells, respectively). Corresponding to this, transcript levels of HoxB4 and Ezh2 did not vary between MPB cells expanded with 5azaD/TSA in the two different cytokine environments. Our studies have the potential to be used to expand HSC from poor mobilizers in order to optimize MPB grafts for transplantation. Disclosures:No relevant conflicts of interest to declare.

Possible Reduced Immunogenicity of Chromatin Modifying Agents Expanded Cord Blood Grafts Is Likely Due to STAT3 Inhibition,

Abstract 4016The suboptimal number of hematopoietic stem cells (HSC) and relatively high incidence of graft failure following umbilical cord blood (CB) transplantation in adult patients remain as major obstacles to the widespread use of CB grafts. Using chromatin modifying agents (CMAs), including 5aza-2-deoxycytidine (5azaD) and trichostatin A (TSA), we have developed a culture strategy that permits 10 to 12 fold expansion of CB CD34+CD90+ cells containing in vivo repopulating cells (Araki et al. Blood 2007, Exp Hematol 2009). It is important that potential ex vivo expansion strategies not increase the capacity of the graft to mount an immune response to an allogeneic host. We have therefore assessed the allo-stimulatory capacity of expanded grafts following 9 days of culture examining immunophenotypic markers of dendritic cell (DC) commitment and also co-stimulatory molecules expressed on CMA-expanded CB cells. 5azaD/TSA-expanded cells co-expressing antigen presenting cell (APC) markers such as HLA-DR/CD86 comprise 17 ± 7% of the graft in contrast to 38 ± 9% in CB cells expanded in control culture and 2 ± 1% of primary CD34+ cells (n=3). Expression of HLA-DR/CD11c was only 3% in 5azaD/TSA-expanded cells in contrast to 37% in CB controls and 60% in primary CD34+ cells. Expression of the co-stimulatory molecule CD40 was not altered. We next performed mixed lymphocyte cultures (MLC), co-culturing irradiated primary or expanded CB cells with allogeneic peripheral blood T cells. The immune stimulatory capacity was measured by thymidine uptake, with CPM values of 3,213 ± 2205, 26,461 ± 670, and 2,859 ± 761 in primary CB cells, CB cells grown in control culture and 5azaD/TSA-expanded cells respectively (control vs. 5azaD/TSA p=0.001, 5azaD/TSA vs D0 p=0.893, n=3). The MLC results suggest that 5azaD/TSA-expanded CB grafts possess 9-fold less allo-stimulatory capacity than control cultures but are similar to primary CB cells. We hypothesized that this reduced allo-stimulatory capacity of 5azaD/TSA-expanded grafts could be due to three possible reasons:1) CMA-induced inhibition of expression of co-stimulatory molecules; 2) inhibition of the differentiation of CD34+ cells to APC; and/or 3) apoptosis of APC due to exposure to CMA. It has been previously shown that activation of the transcription factor STAT3 is indispensable for differentiation of DC from HSC. To study the molecular mechanisms involved in the reduced allo-stimulatory capacity of CMA expanded grafts, transcript levels of several genes, including STAT3 were determined by real time quantitative RT PCR. Interestingly the transcript levels of STAT3 were 2.5-fold higher in 5azaD/TSA-expanded CD34+ cells compared to control cultures. Furthermore the expression of two known inhibitors of STAT3, P21 and GATA1, were upregulated 10.5- and 6.8-fold respectively in 5azaD/TSA-expanded CD34+ cells. Therefore although STAT3 gene expression is increased in 5azaD/TSA-expanded cells it is likely inhibited by the increased levels of the inhibitors P21 and GATA1. In support of this we observed a reduced capacity for DC generation in cultures from 5azaD/TSA-expanded cells compared to control cultures. As expected, a LINE-1 assay, a surrogate marker of global methylation, shows that CpG methylation in 5azaD/TSA-expanded CD34+ cells is reduced to 66% compared to 78% in control cultures. Notably however the decrease in methylation specifically affects CpGs near the promoter site of GATA1 (control 82%, 5azaD/TSA 66%), but not STAT3 or P21. Furthermore, SHP1, a tyrosine phosphatase known to inhibit STAT3 activation, is also upregulated 2-fold in 5azaD/TSA-expanded CD34+ cells compared to control cultures. SHP1 transcription is regulated both by promoter methylation and by interactions with c-MYC, however no appreciable difference in c-MYC expression was detectable between 5azaD/TSA-expanded and control cultures suggesting that SHP1 upregulation in 5azaD/TSA-expanded CD34+ cells also involves promoter hypomethylation. Taken together, our studies indicate that the allo-stimulatory capacity of CB cells expanded in 5azaD/TSA- is reduced rather than increased, likely due to inhibition of an early stage of DC differentiation from CD34+ cells, which is mediated by inhibition of STAT3 activation. Reduced allo-stimulatory capacity of CMA expanded CB grafts may provide benefits by potentially allowing transplantation with less stringent HLA matching. Disclosures:No relevant conflicts of interest to declare.

5.3 R-DHAP Immuno-Chemotherapy is an Effective Remission-Induction Treatment for Fludarabine-Refractory Patients or Those with del17p Before Allogeneic Stem Cell Transplantation; Results of the Planned Interim Analysis of the Dutch-Belgian Prospective Multicenter HOVON88 Trial

tion of PARP inhibitor olaparib and HDAC inhibitor valproic acid (VPA). A strong synergism was observed with the combination of 1-3 M olaparib and micromolar concentrations of VPA. Furthermore, olaparib/VPA treatment could sensitize representative primary CLL tumors with ATM and p53 dysfunction initiated to cycle on a CD40L feeder layer. Consistent with our prediction, western blotting analysis revealed cleavage of caspases and induction of apoptosis in HDAC/PARP inhibitor-treated CLL tumor cells. Furthermore, exposure of ATMand p53-deficient CLL tumor cells to HDAC inhibition delayed kinetics of intranuclear localization of the principal homologous recombination protein Rad51, suggesting attenuating effect of HDAC inhibition on DNA repair. We conclude that combined treatment with the DNA repair inhibitor olaparib and low-toxicity chromatin-modifying agents, HDAC inhibitors, sensitizes ATMand p53-deficient CLL cells by multiple mechanisms and should be considered as a therapeutic approach for this category of aggressive CLL tumors.

Feline Bone Marrow-Derived Mesenchymal Stem Cells Express Several Pluripotent and Neural Markers and Easily Turn into Neural-Like Cells by Manipulation with Chromatin Modifying Agents and Neural Inducing Factors

Recent studies suggest that cellular therapies that utilize mesenchymal stem cells (MSCs), especially ones that have been neurally induced (NI), may provide a functional benefit in a wide range of neurological disorders. Recently, we developed a new method for the efficient generation of neural cells from human bone marrow (BM)-derived MSCs (hMSC). Neural induction was achieved by exposing cells simultaneously to chromatin-modifying agents and neural-inducing factors. When transplanted into injured spinal cords, these NI-hMSCs survived, differentiated, promoted tissue preservation, and significantly improved locomotor recovery of injured animals. In the current study, we sought to determine whether this methodological approach would be equally effective in generating neural-like cells from feline BM-derived MSCs (fMSC). Our long-term goal is to develop an autologous source of neural stem cells that can be used in cellular replacement therapies in large animal (feline) models of neurological disorders. Our results showed that fMSCs exhibited a neural morphology after 48-72 h of neural induction. Immunocytochemistry, ELISA, Western blot, and real-time RT-PCR studies revealed a higher level of expression of several pluripotent and neural genes in NI-fMSCs, the majority of which were expressed in untreated fMSCs at relatively low levels. We concluded that the expression of pluripotency- and neural-associated genes in unmodified fMSCs make them more pliable for reprogramming into a neural fate by manipulation with chromatin modifying agents and neural inducing factors.

Chromatin-modifying agents promote the ex vivo production of functional human erythroid progenitor cells

AbstractPresently, blood transfusion products (TPs) are composed of terminally differentiated cells with a finite life span. We have developed an ex vivo–generated TP composed of erythroid progenitor cells (EPCs) and precursors cells. Several histone deacetylase inhibitors (HDACIs) were used in vitro to promote the preferential differentiation of cord blood (CB) CD34+ cells to EPCs. A combination of cytokines and valproic acid (VPA): (1) promoted the greatest degree of EPC expansion, (2) led to the generation of EPCs which were capable of differentiating into the various stages of erythroid development, (3) led to epigenetic modifications (increased H3 acetylation) of promoters for erythroid-specific genes, which resulted in the acquisition of a gene expression pattern characteristic of primitive erythroid cells, and (4) promoted the generation of a TP that when infused into NOD/SCID mice produced mature RBCs containing both human adult and fetal globins as well Rh blood group Ag which persisted for 3 weeks and the retention of human EPCs and erythroid precursor cells within the BM of recipient mice. This ex vivo–generated EPC-TP likely represents a paradigm shift in transfusion medicine because of its potential to continue to generate additional RBCs after its infusion.

Sequential treatment of CD34+ cells from patients with primary myelofibrosis with chromatin-modifying agents eliminate JAK2V617F-positive NOD/SCID marrow repopulating cells

AbstractBecause primary myelofibrosis (PMF) originates at the level of the pluripotent hematopoietic stem cell (HSC), we examined the effects of various therapeutic agents on the in vitro and in vivo behavior of PMF CD34+ cells. Treatment of PMF CD34+ cells with chromatin-modifying agents (CMAs) but not hydroxyurea, Janus kinase 2 (JAK2) inhibitors, or low doses of interferon-α led to the generation of greater numbers of CD34+ chemokine (C-X-C motif) receptor (CXCR)4+ cells, which were capable of migrating in response to chemokine (C-X-C motif) ligand (CXCL)12 and resulted in a reduction in the proportion of hematopoietic progenitor cells (HPCs) that were JAK2V617F+. Furthermore, sequential treatment of PMF CD34+ cells but not normal CD34+ cells with decitabine (5-aza-2′-deoxycytidine [5azaD]), followed by suberoylanilide hydroxamic acid (SAHA; 5azaD/SAHA), or trichostatin A (5azaD/TSA) resulted in a higher degree of apoptosis. Two to 6 months after the transplantation of CMAs treated JAK2V617F+ PMF CD34+ cells into nonobese diabetic/severe combined immunodeficient (SCID)/IL-2Rγnull mice, the percentage of JAK2V617F/JAK2total in human CD45+ marrow cells was dramatically reduced. These findings suggest that both PMF HPCs, short-term and long-term SCID repopulating cells (SRCs), are JAK2V617F+ and that JAK2V617F+ HPCs and SRCs can be eliminated by sequential treatment with CMAs. Sequential treatment with CMAs, therefore, represents a possible effective means of treating PMF at the level of the malignant SRC.

Valproic Acid Results In Maintenance but Not Expansion of Transplantable Hematopoietic Stem Cells From Human Umbilical Cord Blood

AbstractAbstract 827Epigenetic modifications are considered to be important in determining the fate of hematopoietic stem cells (HSC). We previously demonstrated that the sequential addition of the chromatin-modifying agents (CMA) 5-aza-2′-deoxycytidine (5azaD) and trichostatin A (TSA) expands transplantable HSC (Araki et al. Blood 2007, Exp Hematol 2009). Others have shown that valproic acid (VPA), an HDAC inhibitor, also expands HSC (DeFelice et al. Cancer Res 2005). We thus compared the efficacy of 5azaD/TSA and VPA in promoting the ex vivo expansion of human cord blood (CB) HSC. Cells were incubated with cytokines alone (SCF, Flt3 ligand, TPO and IL-3) or with cytokines and either 5azaD/TSA or VPA, resulting in 2.2-fold, 10.7-fold or 65-fold expansion, respectively, of primitive CD34+CD90+ cells after 9 days (n=3, Cytokine alone vs. VPA p=0.004; Cytokine alone vs. 5azaD/TSA p=0.03; VPA vs. 5azaD/TSA p=0.003). Interestingly, the 10.7-fold expansion of CD34+CD90+ cells following 5azaD/TSA treatment correlated with a 10- and 10.5-fold expansion of short-term colony-forming cells (CFC) and long-term cobblestone area-forming cells (CAFC), respectively. However, the 65-fold expansion of CD34+CD90+ cells achieved with VPA treatment yielded only a 25.6- and 8.4-fold expansion of CFC and CAFC, respectively. These results suggest a marked discordance between the phenotype and function of CD34+CD90+ cells when they are expanded in VPA, but not in 5azaD/TSA. Thus, we examined the in vivo hematopoietic repopulation potential of CMA-expanded CB HSC by quantitating SCID mouse repopulating cells (SRC) using limiting dilution assays. The frequency of SRC was 1 in 22,000 in primary CB cells (n=29 mice), 1 in 123,315, in (cytokine) controls (n=16 mice), 1 in 21,720 with VPA-treatment (n=27 mice), and 1 in 3,147, in 5azaD/TSA-treated CD34+CD90+ cell cultures (n=22 mice). Unlike control, treatment with VPA prevents loss of SRC but only results in SRC maintenance, whereas 5azaD/TSA treatment leads to a 7-fold expansion of SRC. Furthermore, serial transplantation of bone marrow (BM) from primary recipients engrafted with unmanipulated CB cells resulted in engraftment in 2 of 5 secondary mice, while BM from mice engrafted with VPA-treated cells failed to display secondary engraftment (n=5 mice), whereas BM from mice engrafted with 5azaD/TSA-treated cells resulted in engraftment in 5 of 6 secondary mice. Hence, we conclude that treatment of CB CD34+ cells with 5azaD/TSA or VPA results in distinct SRC outcomes-expansion or maintenance, respectively. To dissect the molecular mechanisms that may mediate these distinct SRC fates, we examined genes implicated in HSC self-renewal including HoxB4, Bmi1, STAT3, Ezh2 and PU.1. These gene transcript levels were increased in CD34+ cells treated with either 5azaD/TSA or VPA when compared to control cultures as measured by real time quantitative PCR. In accordance with these studies, CHIP assays using antibody against acetylated histone H4 indicate increased acetylation of the promoters of HoxB4 and Bmi1 genes in both VPA- and 5azaD/TSA-treated cells. In addition, higher levels of HoxB4, Ezh2 and PU.1 proteins were observed in VPA- and 5azaD/TSA-expanded cells, compared to control cultures. Since VPA treatment does not result in SRC expansion, these observations raise questions as to the importance of the upregulation of these genes for HSC expansion. Since the pharmacologic activity of CMAs is short (hours) we hypothesize that temporal effects, including early epigenetic modifications, lead to changes in transcription factor expression, which directly or indirectly promote symmetric or asymmetric divisions ultimately resulting in expansion or maintenance of HSC. Importantly, our global microarray data (n=3) using a human genome affymetrix chip (U133 plus 2.0) revealed a set of differentially expressed genes present in 5azaD/TSA- but not in VPA-expanded CD34+ cells, thus uncovering a potential molecular signature for HSC expansion. Currently, we are examining the molecular interactions of these signature genes and the effects of silencing of these genes on HSC expansion or maintenance which should allow us to begin to unravel the molecular mechanisms involved. In summary our data indicate that treatment of HSCs with different CMAs results in distinct fates: expansion or maintenance of HSC, an observation of potential therapeutic importance. Disclosures:No relevant conflicts of interest to declare.

Chromatin-Modifying Agents for Epigenetic Reprogramming and Endogenous Neural Stem Cell-Mediated Repair in Stroke

The recent explosion of interest in epigenetics and chromatin biology has made a significant impact on our understanding of the pathophysiology of cerebral ischemia and led to the identification of new treatment strategies for stroke, such as those that employ histone deacetylase inhibitors. These are key advances; however, the rapid pace of discovery in chromatin biology and innovation in the development of chromatin-modifying agents implies there are emerging classes of drugs that may also have potential benefits in stroke. Herein, we discuss how various chromatin regulatory factors and their recently identified inhibitors may serve as drug targets and therapeutic agents for stroke, respectively. These factors primarily include members of the repressor element-1 silencing transcription factor (REST)/neuron-restrictive silencer factor macromolecular complex, polycomb group (PcG) proteins, and associated chromatin remodeling factors, which have been linked to the pathophysiology of cerebral ischemia. Further, we suggest that, because of the key roles played by REST, PcG proteins and other chromatin remodeling factors in neural stem and progenitor cell (NSPC) biology, chromatin-modifying agents can be utilized not only to mitigate ischemic injury directly but also potentially to promote endogenous NSPC-mediated brain repair mechanisms.

Inhibition of histone deacetylases by Trichostatin A leads to a HoxB4-independent increase of hematopoietic progenitor/stem cell frequencies as a result of selective survival

DNA and chromatin modifications are critical mediators in the establishment and maintenance of cell type-specific gene expression patterns that constitute cellular identities. One type of modification, the acetylation and deacetylation of histones, occurs reversibly on lysine ε-NH₃(+) groups of core histones via histone acetyl transferases (HAT) and histone deacetylases (HDAC). Hyperacetylated histones are associated with active chromatin domains, whereas hypoacetylated histones are enriched in non-transcribed loci. We analyzed global histone H4 acetylation and HDAC activity levels in mature lineage marker-positive (Lin(+)) and progenitor lineage marker-negative (Lin⁻) hematopoietic cells from murine bone marrow (BM). In addition, we studied the effects of HDAC inhibition on hematopoietic progenitor/stem cell (HPSC) frequencies, cell survival, differentiation and HoxB4 dependence. We observed that Lin⁻ and Lin(+) cells do not differ in global histone H4 acetylation but in HDAC activity levels. Further, we saw that augmented histone acetylation achieved by transient Trichostatin A (TSA) treatment increased the frequency of cells with HPSC immunophenotype and function in the heterogeneous pool of BM cells. Induction of histone hyperacetylation in differentiated BM cells was detrimental, as evidenced by preferential death of mature BM cells upon HDAC inhibition. Finally, TSA treatment of BM cells from HoxB4(-/-) mice revealed that the HDAC inhibitor-mediated increase in HPSC frequencies was independent of HoxB4. Overall, these data indicate the potential of chromatin modifications for the regulation of HPSC. Chromatin-modifying agents may provide potential strategies for ex vivo expansion of HPSC.

Modeling drug sensitivity and resistance in human tumor-derived cell lines

Abstract Accumulating evidence points to the importance of genomic heterogeneity across human tumors as a critical determinant of the variable clinical response to molecularly targeted cancer therapies. We have used cancer cell lines as a model system for examining the relationship between underlying genotypes and sensitivity to candidate anticancer agents. These studies have revealed the utility of large panels of cancer cell lines to capture the genomic heterogeneity of human cancer and to identify clinically relevant genotype-phenotype relationships. These studies have begun to shed light on mechanisms underlying sensitivity as well as de novo and acquired resistance to a variety of small molecule inhibitors. Using this same platform, we have also identified a mechanism of reversible drug “tolerance” that involves a distinct chromatin state displayed transiently by small subpopulations of cancer cells. This drug-tolerant state can be disrupted by chromatin-modifying agents, potentially yielding a therapeutic opportunity to prevent or delay the development of more stable genetically conferred drug resistance mechanisms.

Abstract IA2-1: Modeling drug sensitivity and resistance in human tumor-derived cell lines

Abstract Agccumulating evidence points to the importance of genomic heterogeneity across human tumors as a critical determinant of the variable clinical response to molecularly targeted cancer therapies. We have used cancer cell lines as a model system for examining the relationship between underlying genotypes and sensitivity to candidate anticancer agents. These studies have revealed the utility of large panels of cancer cell lines to capture the genomic heterogeneity of human cancer and to identify clinically relevant genotype- phenotype relationships. These studies have begun to shed light on mechanisms underlying sensitivity as well as de novo and acquired resistance to a variety of small molecule inhibitors. Using this same platform, we have also identified a mechanism of reversible drug tolerance that involves a distinct chromatin state displayed transiently by small subpopulations of cancer cells. This drug-tolerant state can be disrupted by chromatin-modifying agents, potentially yielding a therapeutic opportunity to prevent or delay the development of more stable genetically conferred drug resistance mechanisms. Citation Information: Clin Cancer Res 2010;16(14 Suppl):IA2-1.

A Chromatin-Mediated Reversible Drug-Tolerant State in Cancer Cell Subpopulations

Accumulating evidence implicates heterogeneity within cancer cell populations in the response to stressful exposures, including drug treatments. While modeling the acute response to various anticancer agents in drug-sensitive human tumor cell lines, we consistently detected a small subpopulation of reversibly "drug-tolerant" cells. These cells demonstrate >100-fold reduced drug sensitivity and maintain viability via engagement of IGF-1 receptor signaling and an altered chromatin state that requires the histone demethylase RBP2/KDM5A/Jarid1A. This drug-tolerant phenotype is transiently acquired and relinquished at low frequency by individual cells within the population, implicating the dynamic regulation of phenotypic heterogeneity in drug tolerance. The drug-tolerant subpopulation can be selectively ablated by treatment with IGF-1 receptor inhibitors or chromatin-modifying agents, potentially yielding a therapeutic opportunity. Together, these findings suggest that cancer cell populations employ a dynamic survival strategy in which individual cells transiently assume a reversibly drug-tolerant state to protect the population from eradication by potentially lethal exposures.

Sequential Treatment of CD34+ Cells From Patients with Primary Myelofibrosis with Chromatin Modifying Agents Alters the Behavior of JAK2V617F Positive NOD/SCID Marrow Repopulating Cells.

Abstract 1910Poster Board I-933Primary myelofibrosis (PMF) is likely the consequence of both the acquisition of genetic mutations and epigenetic changes which silence critical genes influencing cell proliferation, differentiation, survival and trafficking. The abnormal trafficking of CD34+ cells in PMF has been attributed to reduced expression of CXCR4 (Shi et al, Cancer Res, 2007; Wang et al, Cancer Res, 2009). We examined the effects of treatment with chromatin modifying agents (CMA), including sequential treatment with a DNA methyltransferase inhibitor, decitabine “5-aza-2'-deoxycytidine (5azaD)”, followed by a histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA) (5azaD/SAHA), or trichostatin A (5azaD/TSA) on the in vitro and in vivo behavior of PMF CD34+ cells (PCC). Sequential treatment with 5azaD/SAHA resulted in a reduction in the number of total cells, CD34+ cells and assayable hematopoietic progenitor cells (HPC) as compared with PCC treated with cytokines alone. By contrast, treatment of PCC with 5azaD/SAHA led to the generation of 2.4-fold greater numbers of CD34+CXCR4+ cells as compared to PCC exposed to cytokines alone and a 15.8-fold increase of the number of CD34+CXCR4+ cells when compared to input PCC (P<0.05). The 5azaD/SAHA treated PCC had a greater migration capacity (20.2±6.9%) toward SDF-1 as compared with PCC exposed to cytokines alone (6.2±2.9%) or primary PCC (2.0±0.5%). These findings suggest that the abnormal trafficking of PCC might be corrected by treatment with 5azaD/SAHA, which is similar to what we had previously reported with 5azaD/TSA. Exposure of PCC to 5azaD/SAHA resulted in a reduction in the proportion of JAK2V617F+ HPCs (p=0.08, 5azaD/SAHA vs cytokines alone; p<0.05, 5azaD/SAHA vs primary PCC) and a reduction in the proportion of JAK2V617F homozygous HPCs in 3 of the 4 patients studied. We next evaluated the effect of sequential treatment with CMA on PMF SCID repopulating cells (SRC) by transplanting primary PCC, PCC treated with cytokines alone, 5azaD/SAHA or 5azaD/TSA and determined the JAK2V617F allelic burden of hCD45+ cells in the marrow of NOD/SCID/IL2Rγnull mice. Two months following transplantation, 2.9% of the marrow cells were hCD45+ cells in mice receiving primary PCC as compared to 0.2% in mice receiving cells treated with cytokines alone or 5azaD/TSA. The engrafted cells in the BM of recipient mice receiving primary PCC were composed of myeloid cells belonging to multiple hematopoietic lineages (CD33+ cells, 2.8%; Glycophorin A+ cells, 0.5%; CD41a+ cells, 0.1% and CD34+ cells, 0.1%) but not CD19+ or CD3+ cells. The JAK2V617F allelic burden of hCD45+ cells isolated from the BM of these mice was analyzed using real-time quantitative PCR. The percentage of JAK2V617F/JAK2total in the gDNA of hCD45+ cells was 71.5% in mice transplanted with primary PCC, 34.7% in mice transplanted with cells treated with cytokines alone and 6.8% in mice transplanted with PCC treated with 5azaD/TSA. Six months following transplantation, a similar degree of human cell chimerism was detected in the BM of recipient mice receiving primary PCC, cells treated with cytokines alone or 5azaD/SAHA. However, the percentage of hCD45+ cells in peripheral blood (PB) of mice transplanted with either primary PCC (0.25%) or PCC treated with cytokines alone (0.1%) was greater than that observed in mice transplanted with PCC treated with 5azaD/SAHA (0.05%). HCD34+ cells were detected in the PB of mice when primary PCC or PCC treated with cytokines alone were transplanted, 2.8% and 1.7%, respectively, but were not detected in mice transplanted with PCC treated with 5azaD/SAHA. These findings suggest that the abnormal trafficking of hCD34+ cells which characterizes PMF, can be recapitulated in NOD/SCID mice. Furthermore, the percentage of JAK2V617F/JAK2total in the gDNA of BM CD45+ cells was 82.5% and 97.6% in mice transplanted with primary PCC as compared to 27.1% and 41.5% in mice transplanted with PCC treated with cytokines alone but was dramatically reduced to 3.6% and 7.4% in mice transplanted with PCC treated with 5azaD/SAHA. These findings suggest that SRC in PMF are JAK2V617F+ and that JAK2V617F+ SRC can be preferentially suppressed by sequential treatment with CMA, leading to engraftment with JAK2V617F- SRC. Sequential treatment with CMA therefore represents a novel means of treating PMF at the level of a JAK2V617F+ SRC, a primitive HPC which shares many properties with the putative PMF stem cell. Disclosures:No relevant conflicts of interest to declare.

A Phase I Study of LBH589, a Novel Histone Deacetylase Inhibitor in Patients with Primary Myelofibrosis (PMF) and Post-Polycythemia/Essential Thrombocythemia Myelofibrosis (Post-PV/ET MF).

Abstract 308 Background:Myelofibrosis (MF) is a myeloproliferative neoplasm characterized by progressive constitutional symptoms, cytopenias, splenomegaly and constitutive mobilization of CD34+ cells. The JAK2V617F mutation is present in approximately 50% of MF patients and the mutational burden has been used as a surrogate marker of disease burden and treatment response. LBH589 (Panobinostat), is a novel histone deacetylase inhibitor (HDACi) with potent inhibitory activity against HDAC and HSP90. CXCR4 has previously shown to be down-regulated in PMF CD34+ cells but can be up-regulated by in vitro treatment with sequential chromatin modifying agents (CMA). In addition, the number of JAK2V617F + progenitor cells (PC) can be reduced by treatment of MF CD34+ cells with CMA. (Shi et al, Cancer Research 67: 6417, 2007) Our working hypothesis is that restoration of CXCR4 expression by PMF CD34+ cells would redirect these cells to the bone marrow niche and reduce extramedullary hematopoiesis and promote elimination of MF PC. In order to test this hypothesis, a series of preclinical studies with purified CD34+ cells isolated from peripheral blood of untreated PMF patients were performed. Cells were cultured with SCF, IL-3, FLT3 and TPO ± LBH589 for 2 days and assayed for total number of cells, CD34+ cells, and % CD34+ cells expressing CXCR4. Both total and CD34+ cell numbers were reduced following LBH589 treatment. CXCR4 expression by cultured CD34+ PMF cells was up-regulated in the presence of LBH589. Cells were placed in semi-solid culture and hematopoietic colonies were enumerated, plucked and genotyped for JAK2V617F. CD34+ cells from 2 of 4 PMF patients treated with LBH589 had a decrease in the number of JAK2V617F homozygous colonies. Based on these data, we are presently conducting an ongoing investigator initiated phase I/II study of LBH589 in patients with MF at our institution. Methods:Patients with Lille class I/II PMF, post-ET/PV MF, collectively referred to as MF were eligible if they had an ANC >1000 and platelet count > 60,000. The primary endpoint of the study is to assess the safety and tolerability of LBH589. Secondary endpoints include measurement of clinical response as assessed by IWG-MRT criteria and assessment of change in JAK2V617F burden, histone acetylation, peripheral blood CD34+ cell number and cytogenetics. DLT was defined as an AE that occurred in the first 28 days on study and judged unrelated to disease progression, intercurrent illness or concomitant medications. AEs were graded by CTCAEv3.0 criteria. LBH589 was given to patients in the first cohort at an oral dose of 20mg TIW and the second cohort at 30mg TIW for 28 days and were eligible to receive additional cycles if no DLT was seen. Results:A total of 12 patients (6 in cohort 1 and 6 in cohort 2) have been enrolled and 8 patients (6 in cohort 1 and 2 in cohort 2) are evaluable to date. Thrombocytopenia is the only DLT observed as of yet and was seen in 2 patients. Grade 3 soft tissue hemorrhage requiring platelet and red blood cell transfusion occurred in a single patient in the first cohort who developed worsening thrombocytopenia from a baseline count of 61,000. In the second cohort at a 30mg TIW dose, Grade 3 thrombocytopenia occurred in 1 patient with a baseline normal platelet count after only 3 doses and resolved within 1 week off the drug. Grade 3 anemia is the only other hematologic AE seen in subsequent cycles in 25% of patients (2/8; a patient in both cohorts). The most common non-hematologic AEs noted were grade 1 nausea, fatigue, diarrhea and musculoskeletal pain. Two patients in cohort 1 have had a greater than 50% reduction in spleen size. One of these patients has also had a significant reduction in RBC transfusion requirements and has complete resolution of splenomegaly at 7 months. By IWG consensus criteria for treatment response, 2 patients (1 JAK2V617F + and 1 -) experienced clinical improvement and 4 patients had stable disease. Conclusion:Treatment of MF patients with LBH589 is associated with acceptable hematologic and non-hematologic toxicity and can result in clinical improvement in patients with advanced MF. Based on these observations, LBH589, an oral HDACi, appears to be a promising new agent for the treatment of both JAK2V617F positive and negative MF. Accrual continues in the phase I portion of this study and data on the effects of LBH589 on a variety of biomarkers including JAK2V617F allele burden, will be presented at the meeting. Disclosures:Off Label Use: LBH589, panobinostat, is a novel oral histone deacetylase inhibitor that is being investigated in myelofibrosis to reverse the splenomegaly and improve cytopenias as well to modulate the JAK2V617F allele burden and potentially change the natural history of the disease. .

Correction of the Abnormal Trafficking of Primary Myelofibrosis CD34+ Cells by Treatment with Chromatin-Modifying Agents

The abnormal trafficking of CD34+ cells is a unique characteristic of primary myelofibrosis (PMF). We have further studied the behavior of PMF CD34+ cells by examining their homing to the marrow and the spleens of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Following the infusion of PMF and normal granulocyte colony-stimulating factor-mobilized peripheral blood (mPB) CD34+ cells into NOD/SCID mice, reduced numbers of PMF CD34+ cells and granulocyte-macrophage colony-forming unit (CFU-GM) compared with mPB were detected in the marrow of these mice, whereas similar numbers of PMF and mPB CD34+ cells and CFU-GM homed to their spleens. The abnormal homing of PMF CD34+ cells was associated with reduced expression of CXCR4, but was not related to the presence of JAK2V617F. The sequential treatment of PMF CD34+ cells with the chromatin-modifying agents 5-aza-2'-deoxycytidine (5azaD) and trichostatin A (TSA), but not treatment with small molecule inhibitors of JAK2, resulted in the generation of increased numbers of CD34+CXCR4+ cells, which was accompanied by enhanced homing of PMF CD34+ cells to the marrow but not the spleens of NOD/SCID mice. Following 5azaD/TSA treatment, JAK2V617F-negative PMF hematopoietic progenitor cells preferentially homed to the marrow but not the spleens of recipient mice. Our data suggest that PMF CD34+ cells are characterized by a reduced ability to home to the marrow but not the spleens of NOD/SCID mice and that this homing defect can be corrected by sequential treatment with chromatin-modifying agents.

Cord blood stem cell expansion is permissive to epigenetic regulation and environmental cues

Augmentation of the number of cord blood (CB) hematopoietic stem cells (HSC) present in a unit is required before it can be considered as an alternative graft for hematopoietic reconstitution for adult patients. In order to further optimize strategies to augment HSC numbers, we examined whether expansion of HSC mediated by epigenetic mechanisms remains permissive to external environmental cues. The chromatin-modifying agents 5-aza-2'-deoxycytidine (5azaD) and trichostatin A (TSA) were used to ameliorate epigenetic alteration of CB cells during ex vivo culture by adding various cytokines. After culture, CD34(+)CD90(+) cell numbers, their division history, in vitro clonogenic potential, and in vivo hematopoietic reconstitution potential and frequency were determined. 5azaD/TSA-treated, CD34(+)CD90(+) cells were greatly influenced in terms of their degree of expansion, clonogenic potential, cell-division rate, and transplantability by the combination of cytokines used in culture. Furthermore, our current results verify that the sequential addition of 5azaD followed by TSA is crucial for expansion of HSC. We demonstrate that following 5azaD/TSA treatment, the rate of CD34(+)CD90(+) cell division is also dependent on the cytokine cocktail and that this is associated with functional changes, including alteration of in vitro clonogenic potential and in vivo reconstitution potential. Our studies indicate there are interactions between intrinsic factors influenced by epigenetic mechanisms and external environmental signals in the regulation of HSC expansion. Epigenetic influences on HSC can be accentuated by environmental factors. Regulation of the rate of divisions may be a critical determinant for the maintenance of HSC functional potency during ex vivo expansion.

Epigenetic Silencing of MicroRNA miR-107 Regulates Cyclin-Dependent Kinase 6 Expression in Pancreatic Cancer

Aberrant expression of microRNAs(miRNAs) has emerged as an important hallmark of cancer. However, the putative mechanisms regulating miRNAs per se are only partially known. It is well established that many tumor suppressor genes in human cancers are silenced by chromatin alterations, including promoter methylation and histone deacet-ylation. We postulated that miRNAs undergo similar epigenetic inactivation in pancreatic cancer. Two human pancreatic cancer cell lines - MiaPACA-2 and PANC-1 - were treated with the demethylating agent, 5-aza-2′-deoxycytidine (5-Aza-dC) or the histone deacetylase inhibitor, trichostatin A, as well as the combination of the two. Expression of miRNAs in control and treated cell lines was assessed using a custom microarray platform. Fourteen miRNAs were upregulated two-fold or greater in each of the cell lines following exposure to both chromatin-modifying agents, including 5 that were in common (miR-107, miR-103, miR-29a, miR-29b, and miR-320) to both MiaPACA-2 and PANC-1. The differential overexpression of miR-107 in the treated cancer cell lines was confirmed by Northern blot assays. Methylation-specific PCR assays for assessment of CpG island methylation status in the 5′ promoter region of the miR-107 primary transcript demonstrated complete loss of methylation upon exposure to 5-Aza-dC. Enforced expression of miR-107 in MiaPACA-2 and PANC-1 cells downregulated in vitro growth, and this was associated with repression of the putative miR-107 target, cyclin-dependent kinase 6, thereby providing a functional basis for the epigenetic inactivation of this miRNA in pancreatic cancer.