Pratik Nagaria

Targeting abnormal DNA repair in therapy-resistant breast cancers

Although hereditary breast cancers have defects in the DNA damage response that result in genomic instability, DNA repair abnormalities in sporadic breast cancers have not been extensively characterized. Recently, we showed that, relative to nontumorigenic breast epithelial MCF10A cells, estrogen receptor–positive (ER+) MCF7 breast cancer cells and progesterone receptor–positive (PR+) MCF7 breast cancer cells have reduced steady-state levels of DNA ligase IV, a component of the major DNA–protein kinase (PK)-dependent nonhomologous end joining (NHEJ) pathway, whereas the steady-state level of DNA ligase IIIα, a component of the highly error-prone alternative NHEJ (ALT NHEJ) pathway, is increased. Here, we show that tamoxifen- and aromatase-resistant derivatives of MCF7 cells and ER−/PR− cells have even higher steady-state levels of DNA ligase IIIα and increased levels of PARP1, another ALT NHEJ component. This results in increased dependence upon microhomology-mediated ALT NHEJ to repair DNA double-strand breaks (DSB) and the accumulation of chromosomal deletions. Notably, therapy-resistant derivatives of MCF7 cells and ER−/PR− cells exhibited significantly increased sensitivity to a combination of PARP and DNA ligase III inhibitors that increased the number of DSBs. Biopsies from ER−/PR− tumors had elevated levels of ALT NHEJ and reduced levels of DNA–PK-dependent NHEJ factors. Thus, our results show that ALT NHEJ is a novel therapeutic target in breast cancers that are resistant to frontline therapies and suggest that changes in NHEJ protein levels may serve as biomarkers to identify tumors that are candidates for this therapeutic approach. Mol Cancer Res; 10(1); 96–107. ©2011 AACR.

DNA double-strand break response in stem cells: mechanisms to maintain genomic integrity.

BACKGROUND Embryonic stem cells (ESCs) represent the point of origin of all cells in a given organism and must protect their genomes from both endogenous and exogenous genotoxic stress. DNA double-strand breaks (DSBs) are one of the most lethal forms of damage, and failure to adequately repair DSBs would not only compromise the ability of SCs to self-renew and differentiate, but will also lead to genomic instability and disease. SCOPE OF REVIEW Herein, we describe the mechanisms by which ESCs respond to DSB-inducing agents such as reactive oxygen species (ROS) and ionizing radiation, compared to somatic cells. We will also discuss whether the DSB response is fully reprogrammed in induced pluripotent stem cells (iPSCs) and the role of the DNA damage response (DDR) in the reprogramming of these cells. MAJOR CONCLUSIONS ESCs have distinct mechanisms to protect themselves against DSBs and oxidative stress compared to somatic cells. The response to damage and stress is crucial for the maintenance of self-renewal and differentiation capacity in SCs. iPSCs appear to reprogram some of the responses to genotoxic stress. However, it remains to be determined if iPSCs also retain some DDR characteristics of the somatic cells of origin. GENERAL SIGNIFICANCE The mechanisms regulating the genomic integrity in ESCs and iPSCs are critical for its safe use in regenerative medicine and may shed light on the pathways and factors that maintain genomic stability, preventing diseases such as cancer. This article is part of a Special Issue entitled Biochemistry of Stem Cells.

Vascular Progenitors From Cord Blood–Derived Induced Pluripotent Stem Cells Possess Augmented Capacity for Regenerating Ischemic Retinal Vasculature

Background— The generation of vascular progenitors (VPs) from human induced pluripotent stem cells (hiPSCs) has great potential for treating vascular disorders such as ischemic retinopathies. However, long-term in vivo engraftment of hiPSC-derived VPs into the retina has not yet been reported. This goal may be limited by the low differentiation yield, greater senescence, and poor proliferation of hiPSC-derived vascular cells. To evaluate the potential of hiPSCs for treating ischemic retinopathies, we generated VPs from a repertoire of viral-integrated and nonintegrated fibroblast and cord blood (CB)–derived hiPSC lines and tested their capacity for homing and engrafting into murine retina in an ischemia-reperfusion model. Methods and Results— VPs from human embryonic stem cells and hiPSCs were generated with an optimized vascular differentiation system. Fluorescence-activated cell sorting purification of human embryoid body cells differentially expressing endothelial/pericytic markers identified a CD31+CD146+ VP population with high vascular potency. Episomal CB-induced pluripotent stem cells (iPSCs) generated these VPs with higher efficiencies than fibroblast-iPSC. Moreover, in contrast to fibroblast-iPSC-VPs, CB-iPSC-VPs maintained expression signatures more comparable to human embryonic stem cell VPs, expressed higher levels of immature vascular markers, demonstrated less culture senescence and sensitivity to DNA damage, and possessed fewer transmitted reprogramming errors. Luciferase transgene-marked VPs from human embryonic stem cells, CB-iPSCs, and fibroblast-iPSCs were injected systemically or directly into the vitreous of retinal ischemia-reperfusion–injured adult nonobese diabetic-severe combined immunodeficient mice. Only human embryonic stem cell– and CB-iPSC–derived VPs reliably homed and engrafted into injured retinal capillaries, with incorporation into damaged vessels for up to 45 days. Conclusions— VPs generated from CB-iPSCs possessed augmented capacity to home, integrate into, and repair damaged retinal vasculature.

Enhancing the Cytotoxic Effects of PARP Inhibitors with DNA Demethylating Agents – A Potential Therapy for Cancer

Poly (ADP-ribose) polymerase inhibitors (PARPis) are clinically effective predominantly for BRCA-mutant tumors. We introduce a mechanism-based strategy to enhance PARPi efficacy based on DNA damage-related binding between DNA methyltransferases (DNMTs) and PARP1. In acute myeloid leukemia (AML) and breast cancer cells, DNMT inhibitors (DNMTis) alone covalently bind DNMTs into DNA and increase PARP1 tightly bound into chromatin. Low doses of DNMTis plus PARPis, versus each drug alone, increase PARPi efficacy, increasing amplitude and retention of PARP1 directly at laser-induced DNA damage sites. This correlates with increased DNA damage, synergistic tumor cytotoxicity, blunting of self-renewal, and strong anti-tumor responses, in vivo in unfavorable AML subtypes and BRCA wild-type breast cancer cells. Our combinatorial approach introduces a strategy to enhance efficacy of PARPis in treating cancer.

Genomic Instability in Chronic Myeloid Leukemia: Targets for Therapy?

Philadelphia positive (Ph+) chronic myeloid leukemia (CML) is characterized by the occurrence of nonrandom genetic and cytogenetic abnormalities during disease progression. Many of these abnormalities are markers for genes which, when altered, can drive the blastic transformation process. Thus, such genetic alterations may be manifestations of an underlying genomic instability resulting from a compromised DNA damage and repair response, leading to advanced stages of CML and resistance to therapy. This article examines the molecular pathways that may lead to genomic instability in CML and the potential of these pathway constituents to be therapeutic targets.

Vascular Progenitors from Cord Blood-Derived iPSC Possess Augmented Capacity for Regenerating Ischemic Retinal Vasculature

Background— The generation of vascular progenitors (VPs) from human induced pluripotent stem cells (hiPSCs) has great potential for treating vascular disorders such as ischemic retinopathies. However, long-term in vivo engraftment of hiPSC-derived VPs into the retina has not yet been reported. This goal may be limited by the low differentiation yield, greater senescence, and poor proliferation of hiPSC-derived vascular cells. To evaluate the potential of hiPSCs for treating ischemic retinopathies, we generated VPs from a repertoire of viral-integrated and nonintegrated fibroblast and cord blood (CB)–derived hiPSC lines and tested their capacity for homing and engrafting into murine retina in an ischemia-reperfusion model. Methods and Results— VPs from human embryonic stem cells and hiPSCs were generated with an optimized vascular differentiation system. Fluorescence-activated cell sorting purification of human embryoid body cells differentially expressing endothelial/pericytic markers identified a CD31+CD146+ VP population with high vascular potency. Episomal CB-induced pluripotent stem cells (iPSCs) generated these VPs with higher efficiencies than fibroblast-iPSC. Moreover, in contrast to fibroblast-iPSC-VPs, CB-iPSC-VPs maintained expression signatures more comparable to human embryonic stem cell VPs, expressed higher levels of immature vascular markers, demonstrated less culture senescence and sensitivity to DNA damage, and possessed fewer transmitted reprogramming errors. Luciferase transgene-marked VPs from human embryonic stem cells, CB-iPSCs, and fibroblast-iPSCs were injected systemically or directly into the vitreous of retinal ischemia-reperfusion–injured adult nonobese diabetic-severe combined immunodeficient mice. Only human embryonic stem cell– and CB-iPSC–derived VPs reliably homed and engrafted into injured retinal capillaries, with incorporation into damaged vessels for up to 45 days. Conclusions— VPs generated from CB-iPSCs possessed augmented capacity to home, integrate into, and repair damaged retinal vasculature.

High-Fidelity Reprogrammed Human IPSCs Have a High Efficacy of DNA Repair and Resemble hESCs in Their MYC Transcriptional Signature.

Human induced pluripotent stem cells (hiPSCs) are reprogrammed from adult or progenitor somatic cells and must make substantial adaptations to ensure genomic stability in order to become “embryonic stem cell- (ESC-) like.” The DNA damage response (DDR) is critical for maintenance of such genomic integrity. Herein, we determined whether cell of origin and reprogramming method influence the DDR of hiPSCs. We demonstrate that hiPSCs derived from cord blood (CB) myeloid progenitors (i.e., CB-iPSC) via an efficient high-fidelity stromal-activated (sa) method closely resembled hESCs in DNA repair gene expression signature and irradiation-induced DDR, relative to hiPSCs generated from CB or fibroblasts via standard methods. Furthermore, sa-CB-iPSCs also more closely resembled hESCs in accuracy of nonhomologous end joining (NHEJ), DNA double-strand break (DSB) repair, and C-MYC transcriptional signatures, relative to standard hiPSCs. Our data suggests that hiPSCs derived via more efficient reprogramming methods possess more hESC-like activated MYC signatures and DDR signaling. Thus, an authentic MYC molecular signature may serve as an important biomarker in characterizing the genomic integrity in hiPSCs.

Alternative Non-homologous End-Joining: Mechanisms and Targeting Strategies in Cancer

Repair of DNA double-strand breaks (DSB)s is essential to the growth and survival of normal as well as cancer cells. Alteration of DSB repair properties in cancer cells can not only drive genomic instability, but also confer increased sensitivity to DSB-inducing agents. Development of agents that selectively inhibit DSB repair pathways will facilitate the design of therapeutic strategies that exploit the differences in DSB repair properties between normal and cancer cells. While mechanisms for classic non-homologous end joining (C-NHEJ) and Homologous recombination (HR) DSB repair pathways have been well studied in cancer, less is known about the alternative and highly error-prone, ALT-NHEJ pathway. Here, we discuss the mechanisms for ALT-NHEJ, alterations in this repair pathway in cancer, inhibition of ALT-NHEJ and future directions for cancer therapies that target this pathway.

Abstract 2848: Combination of DNA methyltransferase and PARP inhibitors as a novel therapy strategy for poor prognosis acute myeloid leukemia

We present here strong preclinical data for a novel, mechanistically based, combinatorial approach to using DNA methyltransferase inhibitors (DNMTi’s), such as decitabine (DAC) and 5-Azacytidine (AzaC), with PARP inhibitors (PARPi’s) as a treatment strategy for acute myelogenous leukemias (AML). AzaC and DAC alone show efficacy in AML but combinatorial approaches will be required to maximize therapeutic responses. PARPi9s have not been well studied as agents for these diseases. The mechanistic rationale for our approach is based upon: 1) data from our group and others showing DNMT9s and PARP co-reside in DNA damage induced protein complexes; 2) the fact that AzaC and DAC trap DNMT9s into DNA via their mechanism of action, led us to hypothesize that these drugs might also increase PARP trapping at DNA damage sites 3) the cytotoxicity of clinically available PARPi’s, and especially the most potent ones, appears to correlate with degree of trapping of PARP1 at DNA damage sites in chromatin. We first find that, indeed, in cultured human AML cells, the DNMTi9s (10 to 20 nM DAC) and PARPi9s (1 to 10 nM BMN 673) alone trap PARP into chromatin and this effect is enhanced when the drugs are combined. Concomitant with this, the combined doses, especially, strongly reduce double strand break (DSB) repair thereby increasing cytotoxic DNA damage. In association with these findings, in methylcellulose cloning assays of both cultured (N = 4) and primary AML cells (N = 9), a combination of the DNMTi9s and PARPi9s strongly decreased colony survival compared to each of the agents alone. Interestingly those cell lines and primary samples expressing poor prognostic FLT3/ITD (Fms-like tyrosine kinase 3 internal tandem duplication) mutations, were particularly sensitive to the combination treatment. Based on all the above results, we developed an in-vivo treatment model, using human FLT3/ITD-positive, MV411-luc xenografts in immunocompromised mice. As opposed to mock treatment, and treatment with AzaC or BMN673, alone, the combined drug treatment, over a 40 day treatment course, starkly and significantly decreases leukemia burden, as measured by luciferase imaging, peripheral blood blast counts and spleen weights. Our data suggest a novel use of both DNMTi9s and PARPi9s in a compelling therapeutic strategy for poor prognosis AML, that will be funded by Van Andel-SU2C for a clinical trial to be based at the University of Maryland. Citation Format: Nidal E. Muvarak, Carine Robert, Pratik K. Nagaria, Khadiza Chowdhury, Eun Yong Choi, Vu Duong, Ashkan Emadi, Maria R. Baer, Rena Lapidus, Stephen Baylin, Feyruz Rassool. Combination of DNA methyltransferase and PARP inhibitors as a novel therapy strategy for poor prognosis acute myeloid leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2848. doi:10.1158/1538-7445.AM2015-2848

Abstract 3859: Histone deacetylase inhibitors promote persistent binding of PARP-1 to DNA double strand breaks in chromatin, thus decreasing repair via non-homologous end joining

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Histone deacetylase inhibitors (HDACi) induce acetylation of histone and non-histone proteins, and trigger an increase in DNA double-strand breaks (DSBs) in addition to widespread acetylation. Additionally, research has also shown that post-translational modifications, such as acetylation/deacetylation of DSB repair proteins, influence their overall function. The focus of our study is to determine how HDACi influence the repair of DSBs by non-homologous end joining (NHEJ) and thus the potential genomic instability of cancer cells. We have previously demonstrated that HDAC inhibition in leukemia cells (K562 and HL60) with trichostatin A (TSA) results in differential acetylation of Ku70 and PARP1, and this is associated with decreased repair via classical NHEJ (c-NHEJ). Our new studies, using a chromosomally integrated reporter for measuring c-NHEJ, confirms that treatments with TSA significantly downregulate c-NHEJ activity. Recent reports have indicated that Ku70 and PARP compete for binding to DSBs following DNA damage. Since the structure of chromatin can impede the accessibility of these repair proteins, we questioned whether HDAC inhibitors directly (via acetylation) or indirectly (via DSB induction), influence the recruitment of repair factors to sites of DNA damage in chromatin. To test this, we performed subcellular fractionation of TSA-treated K562 cells into cytoplasmic, nuclear and chromatin fractions and by Western blotting analysis observed a significantly increased presence of several c-NHEJ proteins (Ku70, Ku80 and 53BP1) and ALT NHEJ protein PARP1. Interestingly, homologous recombination (HR) DSB repair factor RAD51 is also present in chromatin following TSA treatment. Also, chromatin localization of DSB repair proteins increases with higher doses of TSA. To investigate whether TSA treatment alters the binding of these DNA repair proteins to DSBs, we used a chromatin immunoprecipitation (ChIP) assay in K562 cell line with stable chromosomal integration of a c-NHEJ reporter. Here, a DSB can be induced in a site-specific manner and recruitment of DSB repair components can be studied at the site of a DSB in the c-NHEJ reporter. Our ChIP analysis shows that of all DSB repair proteins tested, PARP-1, is most significantly increased and persists at the DSB after TSA treatment. These results suggest that increased binding of PARP1 to DSBs block subsequent repair steps, a phenomenon similar to PARP1 trapping observed with the potent PARP inhibitors. The results presented here provide a mechanism by which HDACi treatment increases cytotoxic DSBs in leukemia cells. Citation Format: Carine Robert, Pratik K. Nagaria, Ivana Gojo, Feyruz Rassool. Histone deacetylase inhibitors promote persistent binding of PARP-1 to DNA double strand breaks in chromatin, thus decreasing repair via non-homologous end joining. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3859. doi:10.1158/1538-7445.AM2015-3859