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The DNA damage response (DDR) pathway is a promising target for anticancer therapies. The androgen receptor and myeloblastosis transcription factors have been reported to regulate expression of an overlapping set of DDR genes in prostate cancer cells. Here, we found that histone demethylase JMJD1A regulates expression of a different set of DDR genes largely through c-Myc. Inhibition of JMJD1A delayed the resolution of γ-H2AX foci, reduced the formation of foci containing ubiquitin, 53BP1, BRCA1 or Rad51, and inhibited the reporter activity of double-strand break (DSB) repair. Mechanistically, JMJD1A regulated expression of DDR genes by increasing not only the level but also the chromatin recruitment of c-Myc through H3K9 demethylation. Further, we found that ubiquitin ligase HUWE1 induced the K27-/K29-linked noncanonical ubiquitination of JMJD1A at lysine-918. Ablation of the JMJD1A noncanonical ubiquitination lowered DDR gene expression, impaired DSB repair, and sensitized response of prostate cells to irradiation, topoisomerase inhibitors or PARP inhibitors. Thus, development of agents that target JMJD1A or its noncanonical ubiquitination may sensitize the response of prostate cancer to radiotherapy and possibly also genotoxic therapy.

A minority of cancers have breast cancer gene (BRCA) mutations that confer sensitivity to poly (ADP-ribose) polymerase (PARP) inhibitors (PARPis), but the role for PARPis in BRCA-proficient cancers is not well established. This suggests the need for novel combination therapies to expand the use of these drugs. Recent reports that low doses of DNA methyltransferase inhibitors (DNMTis) plus PARPis enhance PARPi efficacy in BRCA-proficient AML subtypes, breast, and ovarian cancer open up the possibility that this strategy may apply to other sporadic cancers. We identify a key mechanistic aspect of this combination therapy in nonsmall cell lung cancer (NSCLC): that the DNMTi component creates a BRCAness phenotype through downregulating expression of key homologous recombination and nonhomologous end-joining (NHEJ) genes. Importantly, from a translational perspective, the above changes in DNA repair processes allow our combinatorial PARPi and DNMTi therapy to robustly sensitize NSCLC cells to ionizing radiation in vitro and in vivo. Our combinatorial approach introduces a biomarker strategy and a potential therapy paradigm for treating BRCA-proficient cancers like NSCLC.

The poly(ADP-ribose) polymerase (PARP) family of proteins has been implicated in numerous cellular processes, including DNA repair, translation, transcription, telomere maintenance, and chromatin remodeling. Best characterized is PARP1, which plays a central role in the repair of single strand DNA damage, thus prompting the development of small molecule PARP inhibitors (PARPi) with the intent of potentiating the genotoxic effects of DNA damaging agents such as chemo- and radiotherapy. However, preclinical studies rapidly uncovered tumor-specific cytotoxicity of PARPi in a subset of cancers carrying mutations in the BReast CAncer 1 and 2 genes (BRCA1/2), which are defective in the homologous recombination (HR) DNA repair pathway, and several PARPi are now FDA-approved for single agent treatment in BRCA-mutated tumors. This phenomenon, termed synthetic lethality, has now been demonstrated in tumors harboring a number of repair gene mutations that produce a BRCA-like impairment of HR (also known as a ‘BRCAness’ phenotype). However, BRCA mutations or BRCAness is present in only a small subset of cancers, limiting PARPi therapeutic utility. Fortunately, it is now increasingly recognized that many small molecule agents, targeting a variety of molecular pathways, can induce therapeutic BRCAness as a downstream effect of activity. This review will discuss the potential for targeting a broad range of molecular pathways to therapeutically induce BRCAness and PARPi synthetic lethality.

Histone deacetylase inhibitors (HDACi) induce acetylation of histone and non-histone proteins, and modulate the acetylation of proteins involved in DNA double-strand break (DSB) repair. Non-homologous end-joining (NHEJ) is one of the main pathways for repairing DSBs. Decreased NHEJ activity has been reported with HDACi treatment. However, mechanisms through which these effects are regulated in the context of chromatin are unclear. We show that pan-HDACi, trichostatin A (TSA), causes differential acetylation of DNA repair factors Ku70/Ku80 and poly ADP-ribose polymerase-1 (PARP1), and impairs NHEJ. Repair effects are reversed by treatments with p300/CBP inhibitor C646, with significantly decreased acetylation of PARP1. In keeping with these findings, TSA treatment significantly increases PARP1 binding to DSBs in chromatin. Notably, AML patients treated with HDACi entinostat (MS275) in vivo also show increased formation of poly ADP-ribose (PAR) that co-localizes with DSBs. Further, we demonstrate that PARP1 bound to chromatin increases with duration of TSA exposure, resembling PARP “trapping”. Knockdown of PARP1 inhibits trapping and mitigates HDACi effects on NHEJ. Finally, combination of HDACi with potent PARP inhibitor talazoparib (BMN673) shows a dose-dependent increase in PARP “trapping”, which correlates with increased apoptosis. These results provide a mechanism through which HDACi inhibits deacetylation and increases binding of PARP1 to DSBs, leading to decreased NHEJ and cytotoxicity of leukemia cells.

Reduced NME1 expression in melanoma cell lines, mouse models of melanoma, and melanoma specimens in human patients is associated with increased metastatic activity. Herein, we investigate the role of NME1 in repair of double-stranded breaks (DSBs) and choice of double-strand break repair (DSBR) pathways in melanoma cells. Using chromatin immunoprecipitation, NME1 was shown to be recruited rapidly and directly to DSBs generated by the homing endonuclease I-PpoI. NME1 was recruited to DSBs within 30 min, in concert with recruitment of ataxia-telangiectasia mutated (ATM) protein, an early step in DSBR complex formation, as well as loss of histone 2B. NME1 was detected up to 5 kb from the break site after DSB induction, suggesting a role in extending chromatin reorganization away from the repair site. shRNA-mediated silencing of NME1 expression led to increases in the homologous recombination (HR) and non-homologous end-joining (NHEJ) pathways of double-strand break repair (DSBR), and reduction in the low fidelity, alternative-NHEJ (A-NHEJ) pathway. These findings suggest low expression of NME1 drives DSBR towards higher fidelity pathways, conferring enhanced genomic stability necessary for rapid and error-free proliferation in invasive and metastatic cells. The novel mechanism highlighted in the current study appears likely to impact metastatic potential and therapy-resistance in advanced melanoma and other cancers.

FLT3 internal tandem duplication (ITD) mutations are present in acute myeloid leukemia (AML) in 30% of patients with acute myeloid leukemia (AML), most commonly in those with a normal karyotype, and are associated with short relapse-free survival. Both in vitro and in vivo studies of FLT3 -ITD cell lines have demonstrated reactive oxygen species–mediated DNA double-strand breaks and associated error-prone DNA repair as a mechanism of genomic instability, and we hypothesized that genomic instability might be manifested by cytogenetic changes at relapse of FLT3 -ITD AML. We retrospectively reviewed charts of patients with cytogenetically normal (CN) FLT3- ITD AML treated at the University of Maryland Greenebaum Cancer Center, with attention to metaphase analysis results at relapse. Cytogenetic data were available from first and, when applicable, subsequent relapses for 15 patients diagnosed with CN FLT3 -ITD AML. Among 12 patients with documented FLT3 -ITD at first and, when applicable, subsequent relapse, 10 had cytogenetic changes, including nine with rare structural abnormalities. The high frequency of rare structural chromosome abnormalities at relapse in our case series supports a role of genomic instability in the genesis of relapse, and suggests that reactive oxygen species–generating and DNA repair pathways might be therapeutic targets in FLT3 -ITD AML.

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.

Homologous recombination repair (HRR) protects cells from the lethal effect of spontaneous and therapy-induced DNA double-stand breaks. HRR usually depends on BRCA1/2-RAD51, and RAD52-RAD51 serves as back-up. To target HRR in tumor cells, a phenomenon called "synthetic lethality" was applied, which relies on the addiction of cancer cells to a single DNA repair pathway, whereas normal cells operate 2 or more mechanisms. Using mutagenesis and a peptide aptamer approach, we pinpointed phenylalanine 79 in RAD52 DNA binding domain I (RAD52-phenylalanine 79 [F79]) as a valid target to induce synthetic lethality in BRCA1- and/or BRCA2-deficient leukemias and carcinomas without affecting normal cells and tissues. Targeting RAD52-F79 disrupts the RAD52-DNA interaction, resulting in the accumulation of toxic DNA double-stand breaks in malignant cells, but not in normal counterparts. In addition, abrogation of RAD52-DNA interaction enhanced the antileukemia effect of already-approved drugs. BRCA-deficient status predisposing to RAD52-dependent synthetic lethality could be predicted by genetic abnormalities such as oncogenes BCR-ABL1 and PML-RAR, mutations in BRCA1 and/or BRCA2 genes, and gene expression profiles identifying leukemias displaying low levels of BRCA1 and/or BRCA2. We believe this work may initiate a personalized therapeutic approach in numerous patients with tumors displaying encoded and functional BRCA deficiency.

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.

SummaryReversal of promoter DNA hypermethylation and associated gene silencing is an attractive cancer therapy approach. The DNA methylation inhibitors decitabine and azacitidine are efficacious for hematological neoplasms at lower, less toxic, doses. Experimentally, high doses induce rapid DNA damage and cytotoxicity, which do not explain the prolonged time to response observed in patients. We show that transient exposure of cultured and primary leukemic and epithelial tumor cells to clinically relevant nanomolar doses, without causing immediate cytotoxicity, produce an antitumor “memory” response, including inhibition of subpopulations of cancer stem-like cells. These effects are accompanied by sustained decreases in genomewide promoter DNA methylation, gene reexpression, and antitumor changes in key cellular regulatory pathways. Low-dose decitabine and azacitidine may have broad applicability for cancer management.

The BCL6 oncogenic transcriptional repressor is required for development of germinal center centroblasts, which undergo simultaneous genetic recombination and massive clonal expansion. Although BCL6 is required for survival of centroblasts, its expression in earlier B-cells is toxic. Understanding these opposing effects could provide critical insight into normal B-cell biology and lymphomagenesis. We examined the transcriptional and biological effects of BCL6 in various primary cells. BCL6 repression of ATR was previously shown to play a critical role in the centroblast phenotype. Likewise, we found that BCL6 could impose an ATR-dependent phenotype of attenuated DNA damage sensing and repair in primary fibroblasts and B-cells. BCL6 induced true genomic instability because DNA repair was delayed and was qualitatively impaired, which could be critical for BCL6-induced lymphomagenesis. Although BCL6 can directly repress TP53 in centroblasts, BCL6 induced TP53 expression in primary fibroblasts and B-cells, and these cells underwent p53-dependent growth arrest and senescence in the presence of physiological levels of BCL6. This differential ability to trigger a functional p53 response explains at least in part the different biological response to BCL6 expression in centroblasts versus other cells. The data suggest that targeted re-activation of TP53 could be of therapeutic value in centroblast-derived lymphomas.

DNA amplification plays important roles in the development of drug resistance and tumor progression. One mechanism of DNA amplification involves the breakage-fusion-bridge (BFB) cycle. We previously reported that in Chinese hamster ovary (CHO) cell line, breakage at fragile site 1q31 was associated with mdr1 gene amplification through the BFB mechanism. To elucidate the molecular basis of BFB-mediated DNA amplification, we cloned 1q31 fragile site DNA from a Chinese hamster cell line containing an integrated neomycin-resistance marker. Sequence analyses revealed many characteristics similar to those in other common fragile sites. Moreover, this fragile site contains an evolutionarily conserved novel gene, designated fragile site-associated (FSA) gene. FSA encodes a approximately 16-kb mRNA, from which an unusually large open reading frame (orf) of 5005 amino acids can be deduced. The C-terminal portion of FSA shares a striking sequence similarity to that of Caenorhabditi elegans lipid depleted-3 (lpd-3) gene whose function has been demonstrated to involve in lipid storage. We also demonstrated that expression of FSA is associated with the developmental programs of spermatogenesis and adipogenesis. Our results suggest that the Chinese hamster 1q31 fragile site has many important functions including regulation of mdr1 amplification and differentiation of adipocytes and spermatocytes.

Bloom's syndrome (BS) is an autosomal recessive disorder associated with an elevated incidence of cancers. The gene mutated in BS, BLM, encodes a RecQ helicase family member. BS cells exhibit genomic instability, including excessive homologous recombination and chromosomal aberrations. We reported previously that BS cells also demonstrate increased error-prone nonhomologous endjoining, which could contribute to genomic instability in these cells. Here, we show that BS cells display an abnormality in the timing of replication of both early-replicating genes and late-replicating loci such as chromosomal fragile sites. This delayed replication is associated with a constitutively increased frequency of sites of DNA damage and repair, as determined by the presence of DNA repair factors such as RAD51 and Ku86. In addition, another RecQ family helicase, WRN, also localizes to these repair sites. The presence of these repair sites correlates with the temporal appearance of cyclin B1 expression, indicative of the cells having progressed beyond mid-S phase in the cell division cycle. Critically, these defects in BS cells are the direct result of loss of BLM function, because BS cells phenotypically 'reverted' following transfection with the BLM cDNA no longer show such defects. Thus, our data indicate that constitutive DNA damage is coupled to delayed DNA replication in BS cells.

Aberrant FHIT mRNA transcripts are present in malignant and normal haematopoiesis, but absence of FHIT protein is restricted to leukaemia Alterations of the recently cloned fragile histidine triad (FHIT) gene at chromosome 3p14.2 are frequent in a variety of solid tumours and cancer cell lines. Based on these findings, FHIT has been proposed as a putative tumour-suppressor gene. We evaluated the mRNA expression of the FHIT gene in samples from 55 patients with various haematological malignancies (21 AML, 8 CML, 10 CLL, seven low-grade and nine high-grade Non-Hodgkin's lymphomas), in a panel of 16 leukaemia cell lines, in normal mature haematopoietic cells of both myeloid and lymphoid lineage, as well as in CD34+ haematopoietic progenitor cells. Aberrant FHIT mRNA transcripts were observed in 14/16 (88%) leukaemia cell lines, 43/55 (78%) primary haematological neoplasms, but also in 17/22 (77%) normal controls. 1/16 (6%) cell lines and 7/55 (13%) neoplasms did not express any FHIT mRNA. cDNA sequencing revealed exonic deletions, small DNA insertions and combinations of both. Analysis of genomic DNA showed gene deletions in two myeloid leukaemia cell lines. In contrast to all normal types of haematopoietic cells, FHIT protein was clearly reduced or absent in 8/18 (44%) neoplastic samples tested. Our data indicate that whilst aberrant FHIT mRNA transcripts are seen both in normal and malignant cells, lack of FHIT protein is restricted to leukaemia. Absent FHIT protein expression might contribute to leukaemogenesis.

The myelodysplastic syndromes (MDS) comprise a group of clonal hemopoietic stem cell disorders characterized by ineffective hematopoiesis with an increased propensity to myeloid leukemic (AML) transformation. The underlying molecular basis for MDS and its leukemic evolution is unclear. Except for patients with 17p syndrome, loss of function of the p53 tumor suppressor gene accounts for10% of MDS and AML cases. Recently, mutations of the checkpoint gene, CHK2, the human homologue of the yeast CDS1 and RAD53 genes, have been reported in patients with Li-Fraumeni syndrome who also have normal p53. As p53 mutations are rare in MDS and AML, we investigated the status of the CHK2 gene by reverse transcriptase-polymerase chain reaction (RT-PCR) in patients with MDS (n=10) and patients in whom MDS had transformed into AML (n=3). In the MDS group, we found one patient with a conserved mutation (Lys--Arg) in the forked head-associated (FHA) domain of the CHK2 coding sequence. We also found a deletion in the CHK2 transcript in one patient from the MDS--AML group, resulting in a truncated protein lacking the kinase domain. We conclude that alterations of CHK2 and possible involvement in the pathogenesis of MDS may be a rare event.

A major challenge in cancer treatment is the development of therapies that target cancer cells with little or no toxicity to normal tissues and cells. Alterations in DNA double strand break (DSB) repair in cancer cells include both elevated and reduced levels of key repair proteins and changes in the relative contributions of the various DSB repair pathways. These differences can result in increased sensitivity to DSB-inducing agents and increased genomic instability. The development of agents that selectively inhibit the DSB repair pathways that cancer cells are more dependent upon will facilitate the design of therapeutic strategies that exploit the differences in DSB repair between normal and cancer cells. Here, we discuss the pathways of DSB repair, alterations in DSB repair in cancer, inhibitors of DSB repair and future directions for cancer therapies that target DSB repair.

Disease progression in myeloid malignancies results from the accumulation of "mutations" in genes that control cellular growth and differentiation. Many types of genetic alterations have been identified in myeloid diseases. However, the mechanism(s) by which these cells acquire genetic alterations or "Genomic instability", is less well understood. Increasing evidence suggests that the genetic changes in myeloid malignancies lead to increased production of endogenous sources of DNA damage, such as, reactive oxygen species (ROS). The fusion gene BCR-ABL in chronic myeloid leukemia (CML), FLT3/ITD in acute myeloid leukemia (AML), and RAS mutations in myelodysplastic syndromes (MDS)/myeloproliferative diseases (MPD) result in ROS production. Increased ROS can drive a cycle of genomic instability leading to DNA double strand breaks (DSBs) and altered repair that can lead to acquisition of genomic changes. Evidence is coming to light that defects in a main repair pathway for DSBs, non-homologous end-joining (NHEJ), lead to up-regulation of alternative or "back-up" repair that can create chromosomal deletions and translocations. This article will review evidence for activation of RAS/PI3K/STAT pathways, that lead to increased ROS, DNA damage and defective repair in myeloid diseases, a mechanism for acquisition of additional mutations that can drive disease progression.

Comparative gene mapping analysis in mammals suggests that the major histocompatibility complex (MHC) genes map syntenic with genes, such as glyoxylase 1 (GLO1). In man, the MHC locus and other genes of this syntenic group map to chromosome band 6p21.3, and in mouse, these genes map to chromosome 17. In the hamster, however, only the GLOl gene has been localized; GLO1 maps to chromosome 1, suggesting that the genes within the MHC locus also map to this chromosome. We have localized the hamster MHC class I genes to chromosome band 1q17→q18 by fluorescence in situ hybridization (FISH). These results suggest that GLOl and other syntenic genes also lie within this chromosome region.

A 28-year-old man with Ph-positive chronic granulocytic leukemia (CGL) was treated by high-dose chemoradiotherapy and transplantation of marrow cells harvested from his HLA-identical brother. One year after bone marrow transplantation (BMT) examination of his marrow showed a minority population of Ph-positive cells; their proportion subsequently fell such that 2 years after transplant analysis of marrow cells showed only cytogenetically normal cells. The patient remains clinically normal with a persisting mild lymphocytosis but without hematological evidence of leukemia. We cannot in this patient distinguish between persisting leukemia that later could no longer be recognized and relapse of leukemia that is now suppressed, perhaps only temporarily. This case emphasizes the need for caution in interpreting chromosomal finding after BMT for CGL.