Induced Replication Stress Research Articles

Abstract 383: APOBEC3B is induced by activation of DNA repair pathway and modulates the survival and treatment response in human multiple myeloma

Abstract Constitutive genomic complexity, ongoing DNA damage, and accumulating mutations are observed with progression from monoclonal gammopathy of undetermined significance (MGUS) to active multiple myeloma (MM) to relapsed/refractory disease. Apolipoprotein B mRNA editing catalytic polypeptide-like 3B (APOBEC3B), a DNA cytosine deaminase in cancer mutagenesis, is recently linked to sub-clonal diversification, intra-tumor heterogeneity, and tumor evolution in MM. We here studied the upstream mechanisms of APOBEC3B dysregulation and further defined functional consequences of molecular manipulation of APOBEC3B in MM cells. We first asked whether APOBEC3B levels are altered in MM cells upon treatments with Melphalan (Mel), an alkylating agent used to treat MM which is known to induce replication stress, or with ionizing radiation (IR). Sub-lethal doses of Mel or IR induce APOBEC3B transcript and protein expression in a dose- and time-dependent manner in MM cell lines (n=7), associated with phosphorylation of γH2AX. Significantly, Bortezomib (btz), even at sub-lethal doses triggering DNA damage signaling, induced APOBEC3B expression in multiple MM cell lines. Next, inhibition of ATR or ATM activation pathway significantly decreased Mel- or IR or btz-induced APOBEC3B, suggesting that replication stress induced by Mel, IR, or btz activates ATM/ATR-dependent APOBEC3B induction. We used gene-specific CRISPR knock out (KO), shRNA knockdown (KD), and inducible-shRNA KD to study the functional impact of perturbation of APOBEC3B in MM cells. Both KO and KD of APOBEC3B decreased growth and survival in multiple MM cell lines sensitive or resistant to dexamethasone or lenalidomide. APOBEC3B inhibition significantly enhanced growth arrest followed by apoptosis in MM cells, suggesting that increased APOBEC3B levels contribute to MM cell survival. We next analyzed available data sources for MM cell lines from Cancer Cell Line Encyclopedia and the Genomics of Drug Sensitivity in Cancer, which include microarray gene expression and drug sensitivity information. APOBEC3B expression negatively correlates with MM cell sensitivity to JQ1, a BET inhibitor which inhibit MM cell growth and survival in vitroand in vivo. Importantly, in MM cell lines which are relatively resistant to pomalidomide and JQ1 than other cell lines, APOBEC3B KD by its shRNA enhances sensitivity to both drugs. Taken together, our findings provide new insights into the role of APOBEC3B in triggering cytidine deaminase-induced mutagenesis associated with progression of disease. Furthermore, DNA replication stress triggered by Mel, IR, or btz upregulates APOBEC3B expression, which in turn confers drug resistance. The role of APOBEC in disease pathogenesis and progression, coupled with its role mediating drug resistance, suggest potential utility of targeting APOBEC3B in novel MM therapies. Citation Format: Lijie Xing, Jiye Liu, Liang Lin, Shih-Feng Cho, Kenneth Wen, Tengteng Yu, Gang An, Ligui Qiu, Kenneth Anderson, Yu-Tzu Tai. APOBEC3B is induced by activation of DNA repair pathway and modulates the survival and treatment response in human multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 383.

Sequential Therapy with PARP and WEE1 Inhibitors Minimizes Toxicity while Maintaining Efficacy

We demonstrate that concurrent administration of poly(ADP-ribose) polymerase (PARP) and WEE1 inhibitors is effective in inhibiting tumor growth but poorly tolerated. Concurrent treatment with PARP and WEE1 inhibitors induces replication stress, DNA damage, and abrogates the G2 DNA damage checkpoint in both normal and malignant cells. Following cessation of monotherapy with PARP or WEE1 inhibitors, effects of these inhibitors persist suggesting that sequential administration of PARP and WEE1 inhibitors could maintain efficacy while ameliorating toxicity. Strikingly, while sequential administration mirrored concurrent therapy in cancer cells that have high basal replication stress, low basal replication stress in normal cells protected them from DNA damage and toxicity, thus improving tolerability while preserving efficacy in ovarian cancer xenograft and patient-derived xenograft models.

A phase I/II first-in-human trial of oral SRA737 (a Chk1 inhibitor) given in combination with low-dose gemcitabine in subjects with advanced cancer.

3095 Background: SRA737 is a potent, highly selective and orally-bioavailable inhibitor of checkpoint kinase 1 (Chk1). SRA737-02 was designed to investigate the safety, tolerability and preliminary activity of SRA737 in a novel combination with sub-therapeutic doses of gemcitabine (low dose gemcitabine; LDG) utilized to potentiate SRA737’s activity by induction of replication stress in subjects with advanced solid tumors. Methods: Phase 1 dose escalation investigated cohorts of 3 to 6 subjects receiving escalating doses of SRA737 for 2 days after LDG administration on days 1, 8, 15 of 28-day cycles. Phase 2 expansion cohorts explored the hypothesis that LDG strongly synergizes with SRA737 in subjects with genetically-defined tumors hypothesized to be sensitive to Chk1 inhibition: urothelial, high grade serous ovarian, small cell lung, soft tissue sarcoma, and cervical or anogenital cancers. Results: A total of 55 subjects received SRA737 in 13 dose escalation cohorts at doses of 40 to 600 mg SRA737 combined with LDG doses of 50 to 300 mg/m2. No protocol-defined dose limiting toxicities (DLTs) have been observed. The pharmacokinetic profile of SRA737 revealed an AUC0-24 and Cmax of 3550 ng∙h/mL and 548 ng/mL at 150 mg SRA737. At this dose, the Cmin (52 ng/mL) exceeded that determined in preclinical models to be effective. Enrollment into expansion cohorts was initiated at 500 mg SRA737 plus 100 mg/m2 LDG with intra-patient dose escalation permitted to 250 mg/m2 LDG. Approximately 80 subjects were planned and 82 have been treated. Median treatment duration was 51 days (range 1 to 358). The most common treatment-emergent adverse events were nausea (53%), vomiting (45%), fatigue (40%), diarrhea (38%), and anemia (28%); the majority were of mild to moderate severity. Proof-of-concept clinical activity has been seen in tumor types such as anal, cervical, and rectal. Conclusions: The combination of LDG and SRA737 has been well tolerated. This first-in-human clinical study provides proof-of-concept that sub-therapeutic LDG effectively potentiates SRA737. This novel replication stress-targeted therapy warrants further evaluation in genetically pre-defined solid tumors. Clinical trial information: NCT02797977.

Chromatin remodeler HELLS maintains glioma stem cells through E2F3 and MYC.

Glioblastomas, which contain stem cell-like glioblastoma stem cells (GSCs), are universally lethal cancers. While neural stem cells (NSCs) are usually quiescent, single-cell studies suggest that proliferating glioblastoma cells reside in the GSC population. Interrogating in silico glioma databases for epigenetic regulators that correlate with cell cycle regulation, we identified the chromatin remodeler HELLS as a potential target in glioblastoma. GSCs preferentially expressed HELLS compared with their differentiated tumor progeny and nonmalignant brain cells. Targeting HELLS disrupted GSC proliferation, survival, and self-renewal with induction of replication stress and DNA damage. Investigating potential molecular mechanisms downstream of HELLS revealed that HELLS interacted with the core oncogenic transcription factors, E2F3 and MYC, to regulate gene expression critical to GSC proliferation and maintenance. Supporting the interaction, HELLS expression strongly correlated with targets of E2F3 and MYC transcriptional activity in glioblastoma patients. The potential clinical significance of HELLS was reinforced by improved survival of tumor-bearing mice upon targeting HELLS and poor prognosis of glioma patients with elevated HELLS expression. Collectively, targeting HELLS may permit the functional disruption of the relatively undruggable MYC and E2F3 transcription factors and serve as a novel therapeutic paradigm for glioblastoma.

The Regulation of Replication Stress Response at ALT telomeres by Timeless‐1

All cancer cells need to maintain their telomeres in order to continue proliferating. In 85–90% of all human cancers, maintenance and extension of these telomeres is accomplished by telomerase, which adds a short repetitive sequence to the ends of chromosomes. 10–15% of cancers adopt another method, called the Alternative Lengthening of Telomeres (ALT) pathway. ALT pathway uses homologous recombination to maintain the telomere length of ALT cancers and is vital for the continued growth of ALT cancers. Previously, our lab reported that depletion of FANCM, a key member of the Fanconi Anemia family of genes, induced replication stress response primarily at the ALT telomeres (Pan, PNAS 2017). The objectives of our study were: (1) Elucidate the biological function of human Timeless (Tim1) protein, a very important DNA repair protein, in DNA replication stress response at ALT telomeres. (2) Determine whether depletion of Tim1 affects the viability of ALT cells. To accomplish these goals, we first used siRNA transfection to deplete either Tim1, or FANCM, or both in ALT cells. These cells were then co‐stained with antibodies recognizing TRF2 (a telomere marker) and either BLM (a DNA repair marker) or pRPA (a DNA damage checkpoint marker). The percentage of cells with TRF2 & BLM as well as TRF2 & pRPA foci were quantified. In addition, we also monitored the amount of micronuclei formed in those cells. Formation of micronuclei often represent the unrepaired DNA damages. Finally, cell viability assays of the siRNA transfected cells were done. Cells were allowed to grow for 10 days, fixed and stained with crystal violet. Our results show that: siRNA depletion of FANCM and Tim1 in ALT cells had synergistic/additive foci formation for TRF2 & BLM, and TRF2 & pRPA. siRNA depletion of FANCM and Tim1 in ALT cells had no significant effect on micronuclei formation alone, but increased formation when combined. siRNA depletion of FANCM and Tim1 in ALT cells appears to decrease cell viability to the greatest extent when co‐depleted, as opposed to either one depleted alone. Our findings suggest: (1) ALT cancer cells utilize both FANCM and Tim1 to resolve the replication stress at ALT telomeres; (2) co‐depletion of FANCM and Tim1 is synthetically lethal in ALT cancers. (3) Targeting both FANCM and Tim1 is a potential efficacious strategy in treating ALT cancers.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Lysosome inhibition sensitizes pancreatic cancer to replication stress by aspartate depletion

Functional lysosomes mediate autophagy and macropinocytosis for nutrient acquisition. Pancreatic ductal adenocarcinoma (PDAC) tumors exhibit high basal lysosomal activity, and inhibition of lysosome function suppresses PDAC cell proliferation and tumor growth. However, the codependencies induced by lysosomal inhibition in PDAC have not been systematically explored. We performed a comprehensive pharmacological inhibition screen of the protein kinome and found that replication stress response (RSR) inhibitors were synthetically lethal with chloroquine (CQ) in PDAC cells. CQ treatment reduced de novo nucleotide biosynthesis and induced replication stress. We found that CQ treatment caused mitochondrial dysfunction and depletion of aspartate, an essential precursor for de novo nucleotide synthesis, as an underlying mechanism. Supplementation with aspartate partially rescued the phenotypes induced by CQ. The synergy of CQ and the RSR inhibitor VE-822 was comprehensively validated in both 2D and 3D cultures of PDAC cell lines, a heterotypic spheroid culture with cancer-associated fibroblasts, and in vivo xenograft and syngeneic PDAC mouse models. These results indicate a codependency on functional lysosomes and RSR in PDAC and support the translational potential of the combination of CQ and RSR inhibitors.

DNA polymerase θ (POLQ) is important for repair of DNA double-strand breaks caused by fork collapse

DNA polymerase θ (POLQ) plays an important role in alternative nonhomologous end joining or microhomology-mediated end joining (alt-NHEJ/MMEJ). Here, we show that POLQ is not only required for MMEJ to repair DNA double-strand breaks (DSBs) generated by endonucleases such as I-SceI or Cas9, but is also needed for repair of DSBs derived from DNA nicks generated by Cas9 nickase. Consistently, we found that POLQ deficiency leads to sensitivity to topoisomerase inhibitors that cause DNA single-strand break (SSB) accumulation at replication forks and to ATR inhibitors that induce replication fork collapse. These studies support the function of POLQ in coping with replication stress and repairing DSBs upon fork collapse. POLQ overexpression is present in many cancer types and is associated with poor prognosis, including breast cancer regardless of BRCA1 status. We provide proof-of-concept evidence to support a novel cancer treatment strategy that combines POLQ inhibition with administration of topoisomerase or ATR inhibitors, which induces replication stress and fork collapse. Given the prevalence of POLQ overexpression in tumors, such strategy may have a significant impact on developing targeted cancer treatment.

G-quadruplex DNA drives genomic instability and represents a targetable molecular abnormality in ATRX-deficient malignant glioma

Mutational inactivation of ATRX (α-thalassemia mental retardation X-linked) represents a defining molecular alteration in large subsets of malignant glioma. Yet the pathogenic consequences of ATRX deficiency remain unclear, as do tractable mechanisms for its therapeutic targeting. Here we report that ATRX loss in isogenic glioma model systems induces replication stress and DNA damage by way of G-quadruplex (G4) DNA secondary structure. Moreover, these effects are associated with the acquisition of disease-relevant copy number alterations over time. We then demonstrate, both in vitro and in vivo, that ATRX deficiency selectively enhances DNA damage and cell death following chemical G4 stabilization. Finally, we show that G4 stabilization synergizes with other DNA-damaging therapies, including ionizing radiation, in the ATRX-deficient context. Our findings reveal novel pathogenic mechanisms driven by ATRX deficiency in glioma, while also pointing to tangible strategies for drug development.

APOBEC3B Is Induced By Activation of DNA Repair Pathway and Modulates the Survival and Treatment Response in Human Multiple Myeloma

Constitutive genomic complexity, ongoing DNA damage, and accumulating mutations are observed with progression from monoclonal gammopathy of undetermined significance (MGUS) to active multiple myeloma (MM) to relapsed/refractory disease. Apolipoprotein B mRNA editing catalytic polypeptide-like 3B (APOBEC3B), a DNA cytosine deaminase, plays a prominent role in inducing mutations in multiple human cancers. In MM, APOBEC3B is linked to sub-clonal diversification, intra-tumor heterogeneity, and tumor evolution. Moreover, upregulation of APOBEC3B is associated with poor MM prognosis, suggesting that targeting MM cells with high APOBEC3B may represent a novel therapeutic approach. We here studied the upstream mechanisms of APOBEC3B dysregulation and further defined functional consequences of molecular manipulation of APOBEC3B in MM cells. We characterized its sequelae to identify novel strategies for cancer prevention or treatment by targeting this key driver gene of cancer mutagenesis. Since the expression of APOBEC3B is associated with replication stress in breast cancer, we first asked whether APOBEC3B levels are altered in MM cell lines upon treatments with Melphalan (Mel), an alkylating agent used to treat MM which is known to induce replication stress; or with ionizing radiation (IR). Using quantitative RT-PCR and Western blotting, we found that sub-lethal doses of Mel or IR induce APOBEC3B expression in a dose- and time-dependent manner in MM cell lines (n=7) associated with the phosphorylation of γH2AX. Interestingly bortezomib (btz), even at sub-lethal doses which triggers DNA damage signaling, also induced APOBEC3B expression in H929, MM1S, and U266 MM cells. Since DNA replication stress activates the ATR/ATM pathway, we next investigated whether these kinases mediate APOBEC3B induction following Mel- or IR- or btz-induced DNA replication stress. H929 and MM1S cells were treated with Mel or IR in the presence or absence of ATM or ATR inhibitors, and these cells were then lysed and assayed for APOBEC3B expression. Importantly, inhibition of ATR or ATM activation pathway significantly decreased Mel- or IR or btz-induced APOBEC3B, suggesting that replication stress induced by Mel, IR, or btz, activates transcription of APOBEC3B via an ATM/ATR dependent pathway in vitro. To test the effect of APOBEC3B on cell growth and survival, we used gene-specific CRISPR knock out (KO), shRNA knockdown (KD), and inducible-shRNA KD to study the functional impact of perturbation of APOBEC3B in MM cells. Both KO and KD of APOBEC3B decreased growth and survival in multiple MM cell lines sensitive or resistant to dexamethasone or lenalidomide. Using zombie aqua and annexin V-based flow cytometric analysis, we showed that APOBEC3B inhibition enhanced growth arrest, followed by apoptosis, in these MM cells. These data suggest an important role of increased APOBEC3B levels in MM cell survival. We next analyzed available data sources for MM cell lines from Cancer Cell Line Encyclopedia (CCLE) and the Genomics of Drug Sensitivity in Cancer (GDSC), which include microarray gene expression and drug sensitivity information. APOBEC3B expression negatively correlates with MM cell sensitivity to JQ1, a BET inhibitor which has been reported to inhibit MM cell growth and survival in vitro and in vivo. Importantly, in MM cell lines which are relatively resistant to pomalidomide and JQ1 than other cell lines, APOBEC3B KD by its shRNA enhances sensitivity to both drugs. Taken together, our findings provide new insights into the role of APOBEC3B in triggering cytidine deaminase-induced mutagenesis associated with progression of disease. Furthermore, we show that DNA replication stress triggered by Mel, IR, or btz upregulates APOBEC3B expression, which in turn confers drug resistance. The role of APOBEC in disease pathogenesis and progression, coupled with its role mediating drug resistance, suggest potential utility of targeting APOBEC in novel MM therapies. DisclosuresMunshi:OncoPep: Other: Board of director. Anderson:Bristol Myers Squibb: Consultancy; Millennium Takeda: Consultancy; Celgene: Consultancy; C4 Therapeutics: Equity Ownership, Other: Scientific founder; OncoPep: Equity Ownership, Other: Scientific founder; Gilead: Membership on an entity's Board of Directors or advisory committees.

DNA replication licensing proteins: Saints and sinners in cancer.

DNA replication is all-or-none process in the cell, meaning, once the DNA replication begins it proceeds to completion. Hence, to achieve maximum control of DNA replication, eukaryotic cells employ a multi-subunit initiator protein complex known as "pre-replication complex or DNA replication licensing complex (DNA replication LC). This complex involves multiple proteins which are origin-recognition complex family proteins, cell division cycle-6, chromatin licensing and DNA replication factor 1, and minichromosome maintenance family proteins. Higher-expression of DNA replication LC proteins appears to be an early event during development of cancer since it has been a common hallmark observed in a wide variety of cancers such as oesophageal, laryngeal, pulmonary, mammary, colorectal, renal, urothelial etc. However, the exact mechanisms leading to the abnormally high expression of DNA replication LC have not been clearly deciphered. Increased expression of DNA replication LC leads to licensing and/or firing of multiple origins thereby inducing replication stress and genomic instability. Therapeutic approaches where the reduction in the activity of DNA replication LC was achieved either by siRNA or shRNA techniques, have shown increased sensitivity of cancer cell lines towards the anti-cancer drugs such as cisplatin, 5-Fluorouracil, hydroxyurea etc. Thus, the expression level of DNA replication LC within the cell determines a cell's fate thereby creating a paradox where DNA replication LC acts as both "Saint" and "Sinner". With a potential to increase sensitivity to chemotherapy drugs, DNA replication LC proteins have prospective clinical importance in fighting cancer. Hence, in this review, we will shed light on importance of DNA replication LC with an aim to use DNA replication LC in diagnosis and prognosis of cancer in patients as well as possible therapeutic targets for cancer therapy.

The Combination of the PARP Inhibitor Olaparib and the WEE1 Inhibitor AZD1775 as a New Therapeutic Option for Small Cell Lung Cancer.

Purpose: Introduced in 1987, platinum-based chemotherapy remains standard of care for small cell lung cancer (SCLC), a most aggressive, recalcitrant tumor. Prominent barriers to progress are paucity of tumor tissue to identify drug targets and patient-relevant models to interrogate novel therapies. Following our development of circulating tumor cell patient-derived explants (CDX) as models that faithfully mirror patient disease, here we exploit CDX to examine new therapeutic options for SCLC.Experimental Design: We investigated the efficacy of the PARP inhibitor olaparib alone or in combination with the WEE1 kinase inhibitor AZD1775 in 10 phenotypically distinct SCLC CDX in vivo and/or ex vivo These CDX represent chemosensitive and chemorefractory disease including the first reported paired CDX generated longitudinally before treatment and upon disease progression.Results: There was a heterogeneous depth and duration of response to olaparib/AZD1775 that diminished when tested at disease progression. However, efficacy of this combination consistently exceeded that of cisplatin/etoposide, with cures in one CDX model. Genomic and protein analyses revealed defects in homologous recombination repair genes and oncogenes that induce replication stress (such as MYC family members), predisposed CDX to combined olaparib/AZD1775 sensitivity, although universal predictors of response were not noted.Conclusions: These preclinical data provide a strong rationale to trial this combination in the clinic informed by prevalent, readily accessed circulating tumor cell-based biomarkers. New therapies will be evaluated in SCLC patients after first-line chemotherapy, and our data suggest that the combination of olaparib/AZD1775 should be used as early as possible and before disease relapse. Clin Cancer Res; 24(20); 5153-64. ©2018 AACR.

Cellular senescence induces replication stress with almost no affect on DNA replication timing

ABSTRACT Organismal aging entails a gradual decline of normal physiological functions and a major contributor to this decline is withdrawal of the cell cycle, known as senescence. Senescence can result from telomere diminution leading to a finite number of population doublings, known as replicative senescence (RS), or from oncogene overexpression, as a protective mechanism against cancer. Senescence is associated with large-scale chromatin re-organization and changes in gene expression. Replication stress is a complex phenomenon, defined as the slowing or stalling of replication fork progression and/or DNA synthesis, which has serious implications for genome stability, and consequently in human diseases. Aberrant replication fork structures activate the replication stress response leading to the activation of dormant origins, which is thought to be a safeguard mechanism to complete DNA replication on time. However, the relationship between replicative stress and the changes in the spatiotemporal program of DNA replication in senescence progression remains unclear. Here, we studied the DNA replication program during senescence progression in proliferative and pre-senescent cells from donors of various ages by single DNA fiber combing of replicated DNA, origin mapping by sequencing short nascent strands and genome-wide profiling of replication timing (TRT). We demonstrate that, progression into RS leads to reduced replication fork rates and activation of dormant origins, which are the hallmarks of replication stress. However, with the exception of a delay in RT of the CREB5 gene in all pre-senescent cells, RT was globally unaffected by replication stress during entry into either oncogene-induced or RS. Consequently, we conclude that RT alterations associated with physiological and accelerated aging, do not result from senescence progression. Our results clarify the interplay between senescence, aging and replication programs and demonstrate that RT is largely resistant to replication stress.

Abstract 1372: Novel role of the actin-binding factor Profilin-1 in DNA replication

Abstract Introduction: Proper control of DNA replication is important for genome stability of all mitotic cells, and its deregulation leads to many forms of cancer. While the basic replication machinery and core regulatory factors have been thoroughly studied, new functional regulators continue to emerge. Here we present evidence for a novel function of the actin-binding protein profilin-1 (Pfn1) in DNA replication under both normal and stressed conditions. Pfn1 is essential for actin assembly, cell proliferation and survival, yet showing paradoxical antitumor activities for various types of cancer (breast, bladder, pancreatic, liver). It contains a unique binding site for poly-L-proline (PLP) motifs (XP≥5; X=A, L, I, G, S). Though predominantly cytoplasmic, it is also present in the cellular nucleus with poorly understood functions. Results: We find by chromatin fractionation that Pfn1 knockdown in the untransformed breast epithelial MCF-10A cells significantly inhibits the DNA-binding of many proteins in the replisome complex (e.g. PCNA, MCM-7, RFC, RPA, and DNA polymerase δ and ε) without affecting their cellular level. The knockdown specificity is confirmed by the phenotypic rescue via a Pfn1 construct containing RNAi-resistant silent mutations. In contrast, Pfn1 overexpression increases the DNA-binding of these proteins, and this requires its ability to bind PLPs since a PLP-binding defective Pfn1 mutant (S137D) shows no effect. Interestingly, Pfn1 undergoes robust nuclear translocation upon hydroxyurea (HU)-induced replication stress, and this occurs in various cell lines including MCF-10A and the breast cancer MCF-7 and MDA-MB-231 cells. Consistent with a role of Pfn1 in cellular response to stalled replication forks, its knockdown in HU-treated MCF-10A cells decreases ATR-dependent signaling (p-ATR, p-CHK1, p-P53, p-CDC2, p-RPA) but increases cellular survival, suggesting possible cell cycle checkpoint bypass and evasion of apoptotic cell death in the absence of Pfn1. Conclusions: By uncovering the effects of Pfn1 on DNA replication factors and cellular response to replication stress, we provide for the first time the compelling evidence for a “moonlighting” nuclear function of this well-known actin-binding protein that has been implicated in many forms of cancer. Our data suggest that nuclear Pfn1 is required for normal DNA replication, at least partially through its ability to secure the replisome complex at replication forks. Upon replication stress, more Pfn1 is translocated into the nucleus to facilitate ATR activation and proper DNA damage response (checkpoint activation, DNA repair, and apoptosis) which likely results from Pfn1's ability to stabilize the replication forks and the various associated proteins. Future work will be directed towards understanding the mechanistic details of Pfn1 involvement in DNA replication and translating this novel knowledge into anticancer treatments. Citation Format: Cuige Zhu, Sun-Joong Kim, Sean Holohan, Anna Rogers, Jieya Shao. Novel role of the actin-binding factor Profilin-1 in DNA replication [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1372.

Abstract 3089: YAP and TAZ induce MCM protein expression to facilitate tumor-supporting properties in liver cancer

Abstract Overexpression and nuclear enrichment of the Hippo pathway-regulated transcriptional co-activator Yes-Associated Protein (YAP) is frequently detected in human hepatocellular carcinoma (HCC) and correlates with poor clinical outcome of cancer patients1. In mice, inducible overexpression of the constitutively active isoform YAPS127A induces hepatocellular proliferation leading to hepatomegaly followed by tumor formation within 12-15 weeks. How oncogenic YAP and its paralogue TAZ (syn. WWTR1, WW Domain Containing Transcription Regulator 1) induce uncontrolled proliferation in liver cells is not known, yet. Analysis of expression profiling data, derived from human liver cancer cells after siRNA-mediated inhibition of endogenous YAP, revealed that the Minichromosome Maintenance (MCMs) family members MCM2-7 were positively regulated. MCMs are key components of the pre-replication complex and involved in the formation of the replication fork, which is essential for efficient DNA duplication followed by mitosis2. We confirmed that silencing of YAP and TAZ by independent siRNAs reduced the mRNA and protein expression of MCM2-7 in liver cancer cell lines. In vitro analyses revealed that transcription factors of the TEAD family were the most relevant YAP binding partners needed for the transcriptional regulation of MCMs. Using chromatin immunoprecipitation and Dual Luciferase Reporter Assay, we showed that YAP, TAZ and TEAD4 directly bound the MCM promoter. First in vitro data suggested that viability of liver cancer cells, subjected to drug induced replication stress, was exclusively reduced in cells with reduced YAP, TAZ or MCM protein expression. Administration of the YAP/TAZ/TEAD inhibitor Verteporfin reduced the expression of all MCMs in liver cancer cells. Vice versa, the overexpression of YAPS127A in transgenic mice showed significantly increased levels of the proliferation marker Ki67 and MCM2-7. Expression data from 242 HCC patients illustrated an association between YAP and MCM mRNA levels, with elevated MCMs significantly correlating with worse overall survival and early cancer recurrence3. Immunohistochemical stains of HCC tissue micro-arrays revealed a highly significant correlation between Ki67 expression, nuclear YAP overexpression and nuclear MCM2-7 enrichment. In summary, our results strongly suggest that YAP and TAZ facilitate their tumor-supporting properties through the regulation of MCM2-6, a complete protein complex. We conclude that combined inhibition of YAP and TAZ or perturbation of MCM activity might represent an efficient therapeutic approach for the treatment of a subgroup of HCC patients. 1Tschaharganeh D, et al., Gastroenterol. 2013; 2Deegan TD, et al., Curr Opin Struct Biol. 2016; 3Roessler S, et al., Cancer Res. 2010. Citation Format: Maria Knaub, Sofia Weiler, Stefan Thomann, Peter Schirmacher, Kai Breuhahn. YAP and TAZ induce MCM protein expression to facilitate tumor-supporting properties in liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3089.

Abstract 343: Targeting DNA replication as a therapeutic strategy for acute myeloid leukemia with DNMT3A mutations

Abstract Acute Myeloid Leukemia (AML) is an aggressive malignancy of the blood system, wherein somatic mutations in epigenetic modifier and chromatin remodeling genes are common. Recurrent somatic mutations in the DNA methyltransferase 3 alpha (DNMT3A) gene are detected in about 30% of AML cases, and predict poor outcomes in patients treated with anthracycline-based induction chemotherapy, due to suboptimal response to therapy and subsequent early disease relapse. DNA methylation profiling in both primary AML samples and in animal models carrying a DNMT3A mutation detected modest DNA hypomethylation that did not fully explain how mutant DNMT3A contributed to AML pathogenesis. Recent studies have uncovered disordered nucleosome remodeling in response to DNA topological stress in DNMT3A-mutant cells, which was most pronounced during DNA replication. These changes were accompanied by negative enrichment of the cell cycle-associated gene expression signatures and attenuated CHK1 signaling, implicated in DNA damage response and replication fork integrity. Therefore, we hypothesized that targeting the S-phase in leukemic cells with DNMT3A mutations by pharmacologic agents that stall replication may offer enhanced therapeutic benefit. Indeed, our studies show increased sensitivity to replication stalling pharmacological treatments such as nucleoside analogues cytarabine and fludarabine in different cellular systems harboring DNMT3A mutations. This was accompanied by increased apoptosis as measured by PARP cleavage and annexin V binding.Analysis of the DNA damage signaling revealed elevated CHK1 phosphorylation signaling and accumulation of the DNA damage marker gammaH2A.X. A similar drug sensitivity trend was observed in murine Dnmt3a-mutant bone marrow cells treated with cytarabine, using clonogenic survival in MethoCult as readout. In order to examine this, we are developing isogenic cellular systems with differing DNMT3A mutational status via the CRISPR-Cas9 gene-editing approach. Further, these isogenic cell lines will be used as a tool to characterize the effect of mutant DNMT3A on replication progression, arrest, and resolution under pharmacologically induced replication stress. This will include analysis of the chromatin remodeling at the replication forks, proficiency of DNA damage repair, and dynamic localization of wild-type and mutant DNMT3A within the cells throughout the cell cycle. Finally, we will test the efficacy of replication stress inducing therapies in vivo in a genetically accurate mouse model of Dnmt3a-mutant AML. These studies will shed light on the mechanism of a potential vulnerability in DNMT3A-mutant leukemia cells and may lead to a more effective treatment strategy for this common subtype of AML. Citation Format: Kartika Venugopal, Daphné Dupéré-Richer, Jonathan Licht, Olga Guryanova. Targeting DNA replication as a therapeutic strategy for acute myeloid leukemia with DNMT3A mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 343.

Abstract 2817: RAD6 promotes acquired chemoresistance and a poor prognostic marker in ovarian cancer

Abstract Ovarian cancer (OC) is deadly and incurable for patients who relapse after primary cancer is treated with surgery and platinum-based chemotherapy. Unfortunately, OC has a high rate of disease recurrence (70-80%) and most recurrent tumors are chemoresistant, causing deaths of nearly 15,000 women from this disease each year in U.S.A., which highlights the need for new therapeutic strategies. Platinum drug resistance and refractory disease pose major challenges in treating this disease and are major factors contributing to the poor survival rate of OC patients. Our studies demonstrated that RAD6 (an E2 ubiquitin conjugating enzyme) signaling is stimulated upon chemotherapeutic treatment and promotes expression of DNA repair proteins including Fanconi anemia (FA) pathway. Analysis of clinical samples revealed upregulation of RAD6 in ovarian tumors compared to normal ovarian tissues and RAD6 inhibition reduces cancer cell survival. In a pilot clinical study (n=26) comparing matched OC tumors from patients before and after carboplatin chemotherapy regimen, we found that RAD6 was upregulated after treatment and correlated with both chemoresistance and poor progression-free survival. Knockdown or inhibition of catalytic activity (small-molecule inhibitor) of RAD6 attenuated carboplatin-induced monoubiquitination of target proteins such as FANCD2, PCNA and histone 2B, thereby sensitizing OC cells to carboplatin. Interestingly, inhibition of RAD6 alone in OC cells induced replication stress and reduced cell survival and proliferation by arresting cells in the G2/M phase. Moreover, RAD6 plays an important role in the activation of the trans-lesion synthesis (TLS) pathway by monoubiquitinating PCNA and in the activation of the FA DNA repair pathway. These are critical mechanisms for cells to repair DNA crosslinks induced by platinum drugs. Together with these observations, our data suggest that inhibition of RAD6 could be a viable therapeutic target for overcoming platinum resistance and disease recurrence induced by FA pathway in ovarian cancer. Citation Format: Chinnadurai Mani, David Clark, Ranganatha Somasagara, Sebastian Spencer, Kaushlendra Tripathi, Komariah Palle. RAD6 promotes acquired chemoresistance and a poor prognostic marker in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2817.

Abstract 3376: Early onset of chromosomal instability in breast preneoplasia detected by single-cell genomics

Abstract Chromosomal rearrangements and copy number aberrations are a major mechanism for driver gene alterations in breast cancer. However, the origins of chromosomal instability during breast tumorigenesis are poorly understood. We have recently shown that oncogene expression in normal mammary epithelial cells induces replication stress and a DNA damage response (DDR). The double-strand break sensor Mre11 is required for the oncogene-induced DDR, and suppression of Mre11 is sufficient to promote the development of Her2/Neu-driven breast cancer in a transgenic mouse model. Here we have applied single-cell genomics to primary mammary epithelial cells from this mouse model and demonstrate a profound effect of oncogene expression on chromosomal instability in preneoplastic cells, which is further augmented in Mre11-mutant cells. Her2/Neu-induced chromosomal breakpoints are highly enriched in the vicinity of large genes, suggesting a possible role for gene transcription and/or R-loops in the origin of oncogene-induced genomic fragility. Mre11 hypomorphism does not affect the number of chromosomal breakpoints but alters the genomic aberration signature to one that is over-represented by larger-size deletions. These findings indicate that chromosomal instability is an early event during Her2/Neu-initiated breast neoplasia. Furthermore, DDR perturbation—in the form of Mre11 hypomorphism—is not required for oncogene-induced genomic instability but rather alters the pattern of observed copy number aberrations and promotes the proliferative expansion of genomically unstable clones. Thus, single-cell genomics applied to murine models of breast preneoplasia reveals insights into the origins of chromosomal instability in breast cancer that may inform novel strategies to prognosticate or prevent the progression of high-risk premalignant breast lesions. Citation Format: Katerina Fagan-Solis, Dennis A. Simpson, Luciano Martelotto, Jorge S. Reis-Filho, John H. Petrini, Gaorav P. Gupta. Early onset of chromosomal instability in breast preneoplasia detected by single-cell genomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3376.

Fission Yeast Sirtuin Hst4 Functions in Preserving Genomic Integrity by Regulating Replisome Component Mcl1

The Schizosaccharomyces pombe sirtuin Hst4, functions in the maintenance of genome stability by regulating histone H3 lysine56 acetylation (H3K56ac) and promoting cell survival during replicative stress. However, its molecular function in DNA damage survival is unclear. Here, we show that hst4 deficiency in the fission yeast causes S phase delay and DNA synthesis defects. We identified a novel functional link between hst4 and the replisome component mcl1 in a suppressor screen aimed to identify genes that could restore the slow growth and Methyl methanesulphonate (MMS) sensitivity phenotypes of the hst4Δ mutant. Expression of the replisome component Mcl1 rescues hst4Δ phenotypes. Interestingly, hst4 and mcl1 show an epistatic interaction and suppression of hst4Δ phenotypes by mcl1 is H3K56 acetylation dependent. Furthermore, Hst4 was found to regulate the expression of mcl1. Finally, we show that hSIRT2 depletion results in decreased levels of And-1 (human orthologue of Mcl1), establishing the conservation of this mechanism. Moreover, on induction of replication stress (MMS treatment), Mcl1 levels decrease upon Hst4 down regulation. Our results identify a novel function of Hst4 in regulation of DNA replication that is dependent on H3K56 acetylation. Both SIRT2 and And-1 are deregulated in cancers. Therefore, these findings could be of therapeutic importance in future.

PTEN deletion in luminal cells of mature prostate induces replication stress and senescence in vivo.

Genetic ablation of the tumor suppressor PTEN in prostatic epithelial cells (PECs) induces cell senescence. However, unlike oncogene-induced senescence, no hyperproliferation phase and no signs of DNA damage response (DDR) were observed in PTEN-deficient PECs; PTEN loss-induced senescence (PICS) was reported to be a novel type of cellular senescence. Our study reveals that PTEN ablation in prostatic luminal epithelial cells of adult mice stimulates PEC proliferation, followed by a progressive growth arrest with characteristics of cell senescence. Importantly, we also show that proliferating PTEN-deficient PECs undergo replication stress and mount a DDR leading to p53 stabilization, which is however delayed by Mdm2-mediated p53 down-regulation. Thus, even though PTEN-deficiency induces cellular senescence that restrains tumor progression, as it involves replication stress, strategies promoting PTEN loss-induced senescence are at risk for cancer prevention and therapy.

Mechanisms of Oncogene-Induced Replication Stress: Jigsaw Falling into Place.

Oncogene activation disturbs cellular processes and accommodates a complex landscape of changes in the genome that contribute to genomic instability, which accelerates mutation rates and promotes tumorigenesis. Part of this cellular turmoil involves deregulation of physiologic DNA replication, widely described as replication stress. Oncogene-induced replication stress is an early driver of genomic instability and is attributed to a plethora of factors, most notably aberrant origin firing, replication-transcription collisions, reactive oxygen species, and defective nucleotide metabolism.Significance: Replication stress is a fundamental step and an early driver of tumorigenesis and has been associated with many activated oncogenes. Deciphering the mechanisms that contribute to the replication stress response may provide new avenues for targeted cancer treatment. In this review, we discuss the latest findings on the DNA replication stress response and examine the various mechanisms through which activated oncogenes induce replication stress. Cancer Discov; 8(5); 537-55. ©2018 AACR.