Activating STING1-dependent immune signaling in TP53 mutant and wild-type acute myeloid leukemia.

DNA methyltransferase inhibitors (DNMTis) reexpress hypermethylated genes in cancers and leukemias and also activate endogenous retroviruses (ERVs), leading to interferon (IFN) signaling, in a process known as viral mimicry. In the present study we show that in the subset of acute myeloid leukemias (AMLs) with mutations in TP53, associated with poor prognosis, DNMTis, important drugs for treatment of AML, enable expression of ERVs and IFN and inflammasome signaling in a STING-dependent manner. We previously reported that in solid tumors poly ADP ribose polymerase inhibitors (PARPis) combined with DNMTis to induce an IFN/inflammasome response that is dependent on STING1 and is mechanistically linked to generation of a homologous recombination defect (HRD). We now show that STING1 activity is actually increased in TP53 mutant compared with wild-type (WT) TP53 AML. Moreover, in TP53 mutant AML, STING1-dependent IFN/inflammatory signaling is increased by DNMTi treatment, whereas in AMLs with WT TP53, DNMTis alone have no effect. While combining DNMTis with PARPis increases IFN/inflammatory gene expression in WT TP53 AML cells, signaling induced in TP53 mutant AML is still several-fold higher. Notably, induction of HRD in both TP53 mutant and WT AMLs follows the pattern of STING1-dependent IFN and inflammatory signaling that we have observed with drug treatments. These findings increase our understanding of the mechanisms that underlie DNMTi + PARPi treatment, and also DNMTi combinations with immune therapies, suggesting a personalized approach that statifies by TP53 status, for use of such therapies, including potential immune activation of STING1 in AML and other cancers.

Pharmacologic induction of innate immune signaling directly drives homologous recombination deficiency.

Poly(ADP ribose) polymerase inhibitors (PARPi) have efficacy in triple negative breast (TNBC) and ovarian cancers (OCs) harboring BRCA mutations, generating homologous recombination deficiencies (HRDs). DNA methyltransferase inhibitors (DNMTi) increase PARP trapping and reprogram the DNA damage response to generate HRD, sensitizing BRCA-proficient cancers to PARPi. We now define the mechanisms through which HRD is induced in BRCA-proficient TNBC and OC. DNMTi in combination with PARPi up-regulate broad innate immune and inflammasome-like signaling events, driven in part by stimulator of interferon genes (STING), to unexpectedly directly generate HRD. This inverse relationship between inflammation and DNA repair is critical, not only for the induced phenotype, but also appears as a widespread occurrence in The Cancer Genome Atlas datasets and cancer subtypes. These discerned interactions between inflammation signaling and DNA repair mechanisms now elucidate how epigenetic therapy enhances PARPi efficacy in the setting of BRCA-proficient cancer. This paradigm will be tested in a phase I/II TNBC clinical trial.

DNA methyltransferase inhibitors induce a BRCAness phenotype that sensitizes NSCLC to PARP inhibitor and ionizing radiation.

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.

Personalized synthetic lethality induced by targeting RAD52 in leukemias identified by gene mutation and expression profile.

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.

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.

Defective nonhomologous end joining blocks B-cell development in FLT3/ITD mice.

We have generated an FLT3/ITD knock-in mouse model in which mice with an FLT3/ITD mutation develop myeloproliferative disease (MPD) and a block in early B-lymphocyte development. To elucidate the role of FLT3/ITD signaling in B-cell development, we studied VDJ recombination in the pro-B cells of FLT3/ITD mice and discovered an increased frequency of DNA double strand breaks (DSBs) introduced by the VDJ recombinase. Early pro-B cells from FLT3/ITD mice were found to have a lower efficiency and decreased accuracy of DSB repair by nonhomologous end joining (NHEJ), which is required for rejoining DSBs during VDJ recombination. Reduced NHEJ repair probably results from reduced expression of Ku86, a key component of the classic DNA-PK-dependent NHEJ pathway. In compensation, early pro-B cells from FLT3/ITD cells mice show increased levels of the alternative, and highly error-prone, NHEJ pathway protein PARP1, explaining the increase in repair errors. These data suggest that, in early pro-B cells from FLT3/ITD mice, impairment of classic NHEJ decreases the ability of cells to complete postcleavage DSB ligation, resulting in failure to complete VDJ recombination and subsequent block of B-lymphocyte maturation. These findings might explain the poor prognosis of leukemia patients with constitutive activation of FLT3 signaling.

Pharmacologic Induction of BRCAness in BRCA-Proficient Cancers: Expanding PARP Inhibitor Use.

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.

PARP1 PARylates and stabilizes STAT5 in FLT3-ITD acute myeloid leukemia and other STAT5-activated cancers.

Signal transducer and activator of transcription 5 (STAT5) signaling plays a pathogenic role in both hematologic malignancies and solid tumors. In acute myeloid leukemia (AML), internal tandem duplications of fms-like tyrosine kinase 3 (FLT3-ITD) constitutively activate the FLT3 receptor, producing aberrant STAT5 signaling, driving cell survival and proliferation. Understanding STAT5 regulation may aid development of new treatment strategies in STAT5-activated cancers including FLT3-ITD AML. Poly ADP-ribose polymerase (PARP1), upregulated in FLT3-ITD AML, is primarily known as a DNA repair factor, but also regulates a diverse range of proteins through PARylation. Analysis of STAT5 protein sequence revealed putative PARylation sites and we demonstrate a novel PARP1 interaction and direct PARylation of STAT5 in FLT3-ITD AML. Moreover, PARP1 depletion and PARylation inhibition decreased STAT5 protein expression and activity via increased degradation, suggesting that PARP1 PARylation of STAT5 at least in part potentiates aberrant signaling by stabilizing STAT5 protein in FLT3-ITD AML. Importantly for translational significance, PARPis are cytotoxic in numerous STAT5-activated cancer cells and are synergistic with tyrosine kinase inhibitors (TKI) in both TKI-sensitive and TKI-resistant FLT3-ITD AML. Therefore, PARPi may have therapeutic benefit in STAT5-activated and therapy-resistant leukemias and solid tumors.

Phase I Clinical Trial of DNA Methyltransferase Inhibitor Decitabine and PARP Inhibitor Talazoparib Combination Therapy in Relapsed/Refractory Acute Myeloid Leukemia.

PURPOSE: Patients with acute myeloid leukemia (AML) unfit for, or resistant to, intensive chemotherapy are often treated with DNA methyltransferase inhibitors (DNMTi). Novel combinations may increase efficacy. In addition to demethylating CpG island gene promoter regions, DNMTis enhance PARP1 recruitment and tight binding to chromatin, preventing PARP-mediated DNA repair, downregulating homologous recombination (HR) DNA repair, and sensitizing cells to PARP inhibitor (PARPi). We previously demonstrated DNMTi and PARPi combination efficacy in AML in vitro and in vivo. Here, we report a phase I clinical trial combining the DNMTi decitabine and the PARPi talazoparib in relapsed/refractory AML. PATIENTS AND METHODS: Decitabine and talazoparib doses were escalated using a 3 + 3 design. Pharmacodynamic studies were performed on cycle 1 days 1 (pretreatment), 5 and 8 blood blasts. RESULTS: Doses were escalated in seven cohorts [25 patients, including 22 previously treated with DNMTi(s)] to a recommended phase II dose combination of decitabine 20 mg/m2 intravenously daily for 5 or 10 days and talazoparib 1 mg orally daily for 28 days, in 28-day cycles. Grade 3-5 events included fever in 19 patients and lung infections in 15, attributed to AML. Responses included complete remission with incomplete count recovery in two patients (8%) and hematologic improvement in three. Pharmacodynamic studies showed the expected DNA demethylation, increased PARP trapping in chromatin, increased γH2AX foci, and decreased HR activity in responders. γH2AX foci increased significantly with increasing talazoparib doses combined with 20 mg/m2 decitabine. CONCLUSIONS: Decitabine/talazoparib combination was well tolerated. Expected pharmacodynamic effects occurred, especially in responders.

Targeting abnormal DNA double strand break repair in cancer.

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.

How Epigenetic Therapy Beats Adverse Genetics in Monosomy Karyotype AML.

The study by Greve and colleagues, in this issue of Cancer Research, provides new molecular insights into the intriguing clinical activity of DNA hypomethylating agents (HMA) in patients with acute myeloid leukemia (AML) with monosomal karyotypes. Patients with AML with adverse monosomal karyotypes are known to benefit from HMAs, but not cytarabine, a cytidine analog without HMA activity, but the specific molecular mechanisms remain poorly understood. The authors investigated the mechanistic effects of HMAs on gene reactivation in AML in the context of the most common monosomal karyotypes, genetic deletion of chromosome 7q and 5q. They identified genes with tumor-suppressive properties, an endogenous retrovirus cooperatively repressed by DNA hypermethylation, and increased genetic losses on hemizygous chromosomal regions versus normal biallelic regions in AML cell models. Treatment with HMAs preferentially induced expression of these hemizygous genes to levels similar to those of genes in a biallelic state. In addition to CpG hypomethylation, decitabine treatment resulted in histone acetylation and an open chromatin configuration specifically at hemizygous loci. By using primary blood blasts isolated from patients with AML receiving decitabine and AML patient-derived xenograft models established from patients with either monosomal karyotypes or normal cytogenetics, Greve and colleagues both validated their findings in primary patient samples and demonstrated superior antileukemic activity of decitabine compared with chemotherapy with cytarabine. These mechanistic insights into how epigenetic therapy beats adverse genetics in monosomy karyotype AML will open new therapeutic opportunities for a difficult-to-treat patient group.See related article by Greve et al., p. 834.

Pim kinase inhibitor co-treatment decreases alternative non-homologous end-joining DNA repair and genomic instability induced by topoisomerase 2 inhibitors in cells with FLT3 internal tandem duplication.

Acute myeloid leukemia (AML) with fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) relapses with new chromosome abnormalities following chemotherapy, implicating genomic instability. Error-prone alternative non-homologous end-joining (Alt-NHEJ) DNA double-strand break (DSB) repair is upregulated in FLT3-ITD-expresssing cells, driven by c-Myc. The serine/threonine kinase Pim-1 is upregulated downstream of FLT3-ITD, and inhibiting Pim increases topoisomerase 2 (TOP2) inhibitor chemotherapy drug induction of DNA DSBs and apoptosis. We hypothesized that Pim inhibition increases DNA DSBs by downregulating Alt-NHEJ, also decreasing genomic instability. Alt-NHEJ activity, measured with a green fluorescent reporter construct, increased in FLT3-ITD-transfected Ba/F3-ITD cells treated with TOP2 inhibitors, and this increase was abrogated by Pim kinase inhibitor AZD1208 co-treatment. TOP2 inhibitor and AZD1208 co-treatment downregulated cellular and nuclear expression of c-Myc and Alt-NHEJ repair pathway proteins DNA polymerase θ, DNA ligase 3 and XRCC1 in FLT3-ITD cell lines and AML patient blasts. ALT-NHEJ protein downregulation was preceded by c-Myc downregulation, inhibited by c-Myc overexpression and induced by c-Myc knockdown or inhibition. TOP2 inhibitor treatment increased chromosome breaks in metaphase spreads in FLT3-ITD-expressing cells, and AZD1208 co-treatment abrogated these increases. Thus Pim kinase inhibitor co-treatment both enhances TOP2 inhibitor cytotoxicity and decreases TOP2 inhibitor-induced genomic instability in cells with FLT3-ITD.

Up-regulation of WRN and DNA ligase IIIalpha in chronic myeloid leukemia: consequences for the repair of DNA double-strand breaks.

Expression of oncogenic BCR-ABL in chronic myeloid leukemia (CML) results in increased reactive oxygen species (ROS) that in turn cause increased DNA damage, including DNA double-strand breaks (DSBs). We have previously shown increased error-prone repair of DSBs by nonhomologous end-joining (NHEJ) in CML cells. Recent reports have identified alternative NHEJ pathways that are highly error prone, prompting us to examine the role of the alternative NHEJ pathways in BCR-ABL-positive CML. Importantly, we show that key proteins in the major NHEJ pathway, Artemis and DNA ligase IV, are down-regulated, whereas DNA ligase IIIalpha, and the protein deleted in Werner syndrome, WRN, are up-regulated. DNA ligase IIIalpha and WRN form a complex that is recruited to DSBs in CML cells. Furthermore, "knockdown" of either DNA ligase IIIalpha or WRN leads to increased accumulation of unrepaired DSBs, demonstrating that they contribute to the repair of DSBs. These results indicate that altered DSB repair in CML cells is caused by the increased activity of an alternative NHEJ repair pathway, involving DNA ligase IIIalpha and WRN. We suggest that, although the repair of ROS-induced DSBs by this pathway contributes to the survival of CML cells, the resultant genomic instability drives disease progression.

Reactive oxygen species, DNA damage, and error-prone repair: a model for genomic instability with progression in myeloid leukemia?

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of disorders characterized by ineffective hematopoiesis, with an increased propensity to develop acute myelogenous leukemia (AML). The molecular basis for MDS progression is unknown, but a key element in MDS disease progression is loss of chromosomal material (genomic instability). Using our two-step mouse model for myeloid leukemic disease progression involving overexpression of human mutant NRAS and BCL2 genes, we show that there is a stepwise increase in the frequency of DNA damage leading to an increased frequency of error-prone repair of double-strand breaks (DSB) by nonhomologous end-joining. There is a concomitant increase in reactive oxygen species (ROS) in these transgenic mice with disease progression. Importantly, RAC1, an essential component of the ROS-producing NADPH oxidase, is downstream of RAS, and we show that ROS production in NRAS/BCL2 mice is in part dependent on RAC1 activity. DNA damage and error-prone repair can be decreased or reversed in vivo by N-acetyl cysteine antioxidant treatment. Our data link gene abnormalities to constitutive DNA damage and increased DSB repair errors in vivo and provide a mechanism for an increase in the error rate of DNA repair with MDS disease progression. These data suggest treatment strategies that target RAS/RAC pathways and ROS production in human MDS/AML.

Partnering with PARP inhibitors in acute myeloid leukemia with FLT3-ITD.

Internal tandem duplications within the juxtamembrane domain of fms-like tyrosine kinase 3 (FLT3-ITD) occur in acute myeloid leukemia (AML) cells of 20-25% of patients and are associated with poor treatment outcomes. FLT3 inhibitors have been developed, but have had limited clinical efficacy due to development of resistance, highlighting the need for better understanding of the function of FLT3-ITD and how to target it more effectively using novel combination strategies. Poly (ADP-ribose) polymerase (PARP) inhibitors have shown efficacy in cancers with impaired homologous recombination (HR) due to BRCA mutations, but PARP inhibitor efficacy has not been fully explored in BRCA-proficient cancers, including AML. Recent research has connected inhibition of FLT3-ITD signaling to downregulation of numerous DNA repair proteins, including those involved in HR, and the novel combination with PARP inhibitors induces synthetic lethality in AML. Additionally, PARP inhibitor therapy may also target the highly error-prone alternative non-homologous end-joining (ALT NHEJ) DNA repair pathway in which PARP participates, thereby decreasing genomic instability and development of therapy resistance. Therefore, PARP inhibitors may be attractive therapeutic agents in combination with FLT3 inhibitors in FLT3-ITD AML.

Exploiting epigenetically mediated changes: Acute myeloid leukemia, leukemia stem cells and the bone marrow microenvironment.

Acute myeloid leukemia (AML) derives from the clonal expansion of immature myeloid cells in the bone marrow, and results in the disruption of normal hematopoiesis and subsequent bone marrow failure. The bone marrow microenvironment (BME) and its immune and other supporting cells are regarded to facilitate the survival, differentiation and proliferation of leukemia stem cells (LSCs), which enables AML cells to persist and expand despite treatment. Recent studies have identified epigenetic modifications among AML cells and BME constituents in AML, and have shown that epigenetic therapy can potentially reprogram these alterations. In this review, we summarize the interactions between the BME and LSCs, and discuss changes in how the BME and immune cells interact with AML cells. After describing the epigenetic modifications seen across chromatin, DNA, the BME, and the immune microenvironment, we explore how demethylating agents may reprogram these pathological interactions, and potentially re-sensitize AML cells to treatment.

An Effective Epigenetic-PARP Inhibitor Combination Therapy for Breast and Ovarian Cancers Independent of BRCA Mutations.

Purpose: PARP inhibitors (PARPi) are primarily effective against BRCA1/2-mutated breast and ovarian cancers, but resistance due to reversion of mutated BRCA1/2 and other mechanisms is common. Based on previous reports demonstrating a functional role for DNMT1 in DNA repair and our previous studies demonstrating an ability of DNA methyltransferase inhibitor (DNMTi) to resensitize tumors to primary therapies, we hypothesized that combining a DNMTi with PARPi would sensitize PARPi-resistant breast and ovarian cancers to PARPi therapy, independent of BRCA status.Experimental Design: Breast and ovarian cancer cell lines (BRCA-wild-type/mutant) were treated with PARPi talazoparib and DNMTi guadecitabine. Effects on cell survival, ROS accumulation, and cAMP levels were examined. In vivo, mice bearing either BRCA-proficient breast or ovarian cancer cells were treated with talazoparib and guadecitabine, alone or in combination. Tumor progression, gene expression, and overall survival were analyzed.Results: Combination of guadecitabine and talazoparib synergized to enhance PARPi efficacy, irrespective of BRCA mutation status. Coadministration of guadecitabine with talazoparib increased accumulation of ROS, promoted PARP activation, and further sensitized, in a cAMP/PKA-dependent manner, breast and ovarian cancer cells to PARPi. In addition, DNMTi enhanced PARP "trapping" by talazoparib. Guadecitabine plus talazoparib decreased xenograft tumor growth and increased overall survival in BRCA-proficient high-grade serous ovarian and triple-negative breast cancer models.Conclusions: The novel combination of the next-generation DNMTi guadecitabine and the first-in-class PARPi talazoparib inhibited breast and ovarian cancers harboring either wild-type- or mutant-BRCA, supporting further clinical exploration of this drug combination in PARPi-resistant cancers. Clin Cancer Res; 24(13); 3163-75. ©2018 AACR.

Histone deacetylase inhibitors (HDI) cause DNA damage in leukemia cells: a mechanism for leukemia-specific HDI-dependent apoptosis?

Histone deacetylase inhibitors (HDI) increase gene expression through induction of histone acetylation. However, it remains unclear whether increases in specific gene expression events determine the apoptotic response following HDI administration. Herein, we show that a variety of HDI trigger in hematopoietic cells not only widespread histone acetylation and DNA damage responses but also actual DNA damage, which is significantly increased in leukemic cells compared with normal cells. Thus, increase in H2AX and ataxia telangiectasia mutated (ATM) phosphorylation, early markers of DNA damage, occurs rapidly following HDI administration. Activation of the DNA damage and repair response following HDI treatment is further emphasized by localizing DNA repair proteins to regions of DNA damage. These events are followed by subsequent apoptosis of neoplastic cells but not normal cells. Our data indicate that induction of apoptosis by HDI may result predominantly through accumulation of excessive DNA damage in leukemia cells, leading to activation of apoptosis.

CHD4 Has Oncogenic Functions in Initiating and Maintaining Epigenetic Suppression of Multiple Tumor Suppressor Genes.

An oncogenic role for CHD4, a NuRD component, is defined for initiating and supporting tumor suppressor gene (TSG) silencing in human colorectal cancer. CHD4 recruits repressive chromatin proteins to sites of DNA damage repair, including DNA methyltransferases where it imposes de novo DNA methylation. At TSGs, CHD4 retention helps maintain DNA hypermethylation-associated transcriptional silencing. CHD4 is recruited by the excision repair protein OGG1 for oxidative damage to interact with the damage-induced base 8-hydroxydeoxyguanosine (8-OHdG), while ZMYND8 recruits it to double-strand breaks. CHD4 knockdown activates silenced TSGs, revealing their role for blunting colorectal cancer cell proliferation, invasion, and metastases. High CHD4 and 8-OHdG levels plus low expression of TSGs strongly correlates with early disease recurrence and decreased overall survival.

Molecular cloning of Chinese hamster 1q31 chromosomal fragile site DNA that is important to mdr1 gene amplification reveals a novel gene whose expression is associated with spermatocyte and adipocyte differentiation.

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.