Melissa Singh

Regulation of DNA Repair through DeSUMOylation and SUMOylation of Replication Protein A Complex

The replication protein A complex (RPA) plays a crucial role in DNA replication and damage response. However, it is not known whether this complex is regulated by the SUMOylation pathway. Here, we show that the 70 kDa subunit of RPA (RPA70) associates with a Sentrin/SUMO-specific protease, SENP6, in the nucleus to maintain RPA70 in a hypoSUMOylated state during S phase. Campothecin (CPT), an inducer of replication stress, dissociates SENP6 from RPA70, allowing RPA70 to be modified by a small ubiquitin-like modifier 2/3 (SUMO-2/3). RPA70 SUMOylation facilitates recruitment of Rad51 to the DNA damage foci to initiate DNA repair through homologous recombination (HR). Cell lines that expressed a RPA70 mutant that cannot be SUMOylated are defective in HR and have a marked increase in sensitivity to CPT. These results demonstrate that SUMOylation status of RPA70 plays a critical role in the regulation of DNA repair through homologous recombination.

Inhibition of LSD1 sensitizes glioblastoma cells to histone deacetylase inhibitors

Glioblastoma multiforme (GBM) is a particularly aggressive brain tumor and remains a clinically devastating disease. Despite innovative therapies for the treatment of GBM, there has been no significant increase in patient survival over the past decade. Enzymes that control epigenetic alterations are of considerable interest as targets for cancer therapy because of their critical roles in cellular processes that lead to oncogenesis. Several inhibitors of histone deacetylases (HDACs) have been developed and tested in GBM with moderate success. We found that treatment of GBM cells with HDAC inhibitors caused the accumulation of histone methylation, a modification removed by the lysine specific demethylase 1 (LSD1). This led us to examine the effects of simultaneously inhibiting HDACs and LSD1 as a potential combination therapy. We evaluated induction of apoptosis in GBM cell lines after combined inhibition of LSD1 and HDACs. LSD1 was inhibited by targeted short hairpin RNA or pharmacological means and inhibition of HDACs was achieved by treatment with either vorinostat or PCI-24781. Caspase-dependent apoptosis was significantly increased (>2-fold) in LSD1-knockdown GBM cells treated with HDAC inhibitors. Moreover, pharmacologically inhibiting LSD1 with the monoamine oxidase inhibitor tranylcypromine, in combination with HDAC inhibitors, led to synergistic apoptotic cell death in GBM cells; this did not occur in normal human astrocytes. Taken together, these results indicate that LSD1 and HDACs cooperate to regulate key pathways of cell death in GBM cell lines but not in normal counterparts, and they validate the combined use of LSD1 and HDAC inhibitors as a therapeutic approach for GBM.

Preclinical activity of combined HDAC and KDM1A inhibition in glioblastoma.

BACKGROUND Glioblastoma (GBM) is the most common and aggressive form of brain cancer. Our previous studies demonstrated that combined inhibition of HDAC and KDM1A increases apoptotic cell death in vitro. However, whether this combination also increases death of the glioma stem cell (GSC) population or has an effect in vivo is yet to be determined. Therefore, we evaluated the translational potential of combined HDAC and KDM1A inhibition on patient-derived GSCs and xenograft GBM mouse models. We also investigated the changes in transcriptional programing induced by the combination in an effort to understand the induced molecular mechanisms of GBM cell death. METHODS Patient-derived GSCs were treated with the combination of vorinostat, a pan-HDAC inhibitor, and tranylcypromine, a KDM1A inhibitor, and viability was measured. To characterize transcriptional profiles associated with cell death, we used RNA-Seq and validated gene changes by RT-qPCR and protein expression via Western blot. Apoptosis was measured using DNA fragmentation assays. Orthotopic xenograft studies were conducted to evaluate the effects of the combination on tumorigenesis and to validate gene changes in vivo. RESULTS The combination of vorinostat and tranylcypromine reduced GSC viability and displayed efficacy in the U87 xenograft model. Additionally, the combination led to changes in apoptosis-related genes, particularly TP53 and TP73 in vitro and in vivo. CONCLUSIONS These data support targeting HDACs and KDM1A in combination as a strategy for GBM and identifies TP53 and TP73 as being altered in response to treatment.

Expression and Activity of Fyn Mediate Proliferation and Blastic Features of Chronic Myelogenous Leukemia

The BCR-ABL1 oncogene is a tyrosine kinase that activates many signaling pathways, resulting in the induction of chronic myeloid leukemia (CML). Kinase inhibitors, such as imatinib, have been developed for the treatment of CML; however, the terminal, blast crisis phase of the disease remains a clinical challenge. Blast crisis CML is difficult to treat due to resistance to tyrosine kinase inhibitors, increased genomic instability and acquired secondary mutations. Our recent studies uncovered a role for Fyn in promoting BCR-ABL1 mediated cell growth and sensitivity to imatinib. Here we demonstrate that Fyn contributes to BCR-ABL1 induced genomic instability, a feature of blast crisis CML. Bone marrow cells and mouse embryonic fibroblasts derived from Fyn knockout mice transduced with BCR-ABL1 display slowed growth and clonogenic potential as compared to Fyn wild-type BCR-ABL1 expressing counterparts. K562 cells overexpressing constitutively active Fyn kinase were larger in size and displayed an accumulation of genomic abnormalities such as chromosomal aberrations and polyploidy. Importantly, loss of Fyn protected mouse embryonic fibroblast cells from increased number of chromosomal aberrations and fragments induced by BCR-ABL1. Together, these results reveal a novel role for Fyn in regulating events required for genomic maintenance and suggest that Fyn kinase activity plays a role in the progression of CML to blast crisis.

Inhibition of the NADPH oxidase regulates heme oxygenase 1 expression in chronic myeloid leukemia

Patients with chronic myelogenous leukemia (CML) in blast crisis have a poor response to tyrosine kinase inhibitors designed to inhibit the breakpoint cluster region–v‐Abelson murine leukemia viral oncogene homolog 1 (BCR‐ABL1) oncogene. Recent work has demonstrated that heme oxygenase 1 (HO‐1) expression is increased in BCR‐ABL1–expressing cells and that the inhibition of HO‐1 in CML leads to reduced cellular growth, suggesting that HO‐1 may be a plausible target for therapy. The objective of the current study was to clarify the mechanism of HO‐1 overexpression and the role of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase as a contributor to this mechanism in CML.

Induction of cell death by the novel proteasome inhibitor marizomib in glioblastoma in vitro and in vivo

New therapies for glioblastoma (GBM) are needed, as five-year survival is <10%. The proteasome inhibitor marizomib (MRZ) has inhibitory and death-inducing properties unique from previous inhibitors such as bortezomib (BTZ), and has not been well examined in GBM. We evaluated the mechanism of death and in vivo properties of MRZ in GBM. The activation kinetics of initiator caspases 2, 8, and 9 were assessed using chemical and knockdown strategies to determine their contribution to cell death. Blood brain barrier permeance and proteasome inhibition by MRZ and BTZ were examined in an orthotopic GBM model. Blockade of caspase 9, relative to other caspases, was most protective against both MRZ and BTZ. Only MRZ increased the proteasome substrate p27 in orthotopic brain tumors after a single injection, while both MRZ and BTZ increased p21 levels after multiple treatments. Cleavage of caspase substrate lamin A was increased in orthotopic brain tumors from mice treated with MRZ or BTZ and the histone deacetylase inhibitor vorinostat. Our data indicate that MRZ induces caspase 9-dependent death in GBM, suggesting drug efficacy biomarkers and possible resistance mechanisms. MRZ reaches orthotopic brain tumors where it inhibits proteasome function and increases death in combination with vorinostat.

Abstract B49: Mechanism of cell death induction by dual targeting of HDACs and lysine specific demethylase, LSD1, in leukemias and brain tumors

Targeting of histone modifications is clinically feasible due to the development of histone deacetylase inhibitors (HDACi), several of which are FDA-approved. In order to identify strategies to augment activity of HDACi, we tested inhibition of lysine specific demethylase-1 (KDM1A/LSD1) in combination with HDACi in malignancies where single agent activity was limited in clinical trials: acute leukemia and glioblastoma, In brain tumor cell lines, patient derived glioblastoma stem cells, and a series of acute leukemia lines, we find high expression of LSD1 protein. Using either knockdown of LSD1, or chemical inhibition via the FDA approved drug, tranylcypromine, we report significant enhancement of HDACi induced cell death. Multiple HDACi were used including vorinostat, panobinostat and entinostat, and cytoxicity was consistently heightened when combined with LSD1 inhibition. Indicative of selectivity of this therapeutic approach, augmentation of HDACi cytotoxicity occurred exclusively in tumor cells, and not in normal counterparts. Caspase activation is a feature of cell death induction by HDAC and LSD1 inhibition and pan-caspase inhibition blocks cell death. Using chemical inhibitors of caspase-8, -9 and -3 and caspase-8 deficient cells, we demonstrate a reliance upon extrinsic pathways of caspase activation by the combination therapy. However, data obtained with caspase-8 deficient cells showed a lack of protection in cells treated with LSD1 inhibitor combined with pan-HDACi (vorinostat and panobinostat), whereas there was significant protection in cells treated with entinostat (a Class I HDACi) and LSD1 inhibitor. This suggests that pan-HDACi may trigger alternate caspases, such as caspase-10, which are blocked by peptide inhibitors but are still active in caspase-8 deficient cells. To gain additional insight into the contribution of LSD1 to cytotoxicity, an apoptosis focused array and RNA-Seq were conducted. The majority of gene expression changes were seen in cells exposed to HDACi alone. However, some of these gene expression changes were further enhanced when LSD1 was also inhibited. Expression of the tumor suppressor genes, p53 and p73, was significantly reduced by HDACi alone, and by the combination of HDACi and LSD1 inhibition. Hrk, a pro-apoptotic Bcl-2 family member, known to be silenced in glioblastoma, was re-expressed by the combination therapy. Time course analyses indicated the loss of p53, p73 and increased Hrk, was an early event, occurring within 6 hours of exposure to HDACi and LSD1 inhibitors, and preceded caspase activation. Upregulation of Hrk transcript was robust and rapid. We observed a ten-fold increase within 3 h of exposure to these agents. Further studies will evaluate the contribution of these early changes to caspase activation, thereby delineating the pathway by which HDACs and LSD1 can be optimally targeted in leukemia and brain tumor model systems. Citation Format: Melissa Singh, Hayley Donnella, Lorimar Ramirez, Joya Chandra. Mechanism of cell death induction by dual targeting of HDACs and lysine specific demethylase, LSD1, in leukemias and brain tumors. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B49.

Inhibition of the NADPH oxidase regulates heme oxygenase 1 expression in chronic myeloid leukemia: Nox2 Influences HO-1 Expression in CML

Patients with chronic myelogenous leukemia (CML) in blast crisis have a poor response to tyrosine kinase inhibitors designed to inhibit the breakpoint cluster region–v‐Abelson murine leukemia viral oncogene homolog 1 (BCR‐ABL1) oncogene. Recent work has demonstrated that heme oxygenase 1 (HO‐1) expression is increased in BCR‐ABL1–expressing cells and that the inhibition of HO‐1 in CML leads to reduced cellular growth, suggesting that HO‐1 may be a plausible target for therapy. The objective of the current study was to clarify the mechanism of HO‐1 overexpression and the role of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase as a contributor to this mechanism in CML.

Inhibition of the NADPH oxidase regulates HO-1 expression in chronic myeloid leukemia

Patients with chronic myelogenous leukemia (CML) in blast crisis have a poor response to tyrosine kinase inhibitors designed to inhibit the breakpoint cluster region–v‐Abelson murine leukemia viral oncogene homolog 1 (BCR‐ABL1) oncogene. Recent work has demonstrated that heme oxygenase 1 (HO‐1) expression is increased in BCR‐ABL1–expressing cells and that the inhibition of HO‐1 in CML leads to reduced cellular growth, suggesting that HO‐1 may be a plausible target for therapy. The objective of the current study was to clarify the mechanism of HO‐1 overexpression and the role of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase as a contributor to this mechanism in CML.

Abstract 1627: Requirement for the Src family kinase, Fyn, in proliferation of BCR-ABL1 transduced bone marrow and aneuploidy in CML

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Oncogenic signals stemming from the BCR/ABL kinase drive the development and progression of chronic myeloid leukemia (CML). Hence, therapies blocking kinase activity have proven extremely successful at managing this hematological malignancy. However, treatment of the terminal and progressed state of CML, blast crisis, remains difficult. Previous work from our group has identified the Src family kinase, Fyn, as being upregulated in blast crisis CML. We also reported that knockdown of Fyn slowed CML growth in vitro and in vivo and sensitized cells to BCR-ABL1 kinase inhibition with imatinib. Recent work using microarray profiling of kinase inhibitor resistant cells from a separate group has shown that imatinib resistance is associated with Fyn expression. In the current study, we utilized bone marrow cells from Fyn deficient mice to test the requirement for Fyn in p210 BCR-ABL1 mediated survival. Bone marrow cells from Fyn knockout and Fyn wildtype mice were harvested and transduced with a GFP expressing retroviral construct (MigR1) or MigR1-p210 BCR-ABL1. Cells were sorted for GFP expression and BCR-ABL1 expression was confirmed by Western blotting. Cell growth in culture was monitored over the course of three weeks in Fyn deficient and wildtype cells expressing BCR-ABL1. Fourteen days post sorting, the number of cells lacking Fyn were more than 50% lower than Fyn wildtype cells transduced with BCR-ABL1. Clonogenic assays further indicated that Fyn significantly promotes BCR-ABL1 induced bone marrow cell growth. Colony numbers in Fyn deficient cells expressing BCR-ABL1 were less than half that seen in Fyn wildtype cells. To address the role for Fyn kinase activity in these effects, constitutively active and dominant negative Fyn constructs have been transduced into BCR-ABL1 expressing cells. Constitutively active Fyn caused a high degree of chromosomal aberrations, fusions and aneuploidy suggestive of genomic instability. Taken together, these data highlight a significant role for Fyn in BCR-ABL1 mediated proliferation and secondary chromosomal aberrations, which are both features characteristic of blast crisis CML. Our results indicate that strategies to inhibit Fyn expression and kinase activity will be therapeutically relevant for progressed and/or refractory CML patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1627.