The famous French scientist, Emile Roux, was previously discovered to have been secretly married to an English woman, Rose Anna Shedlock, one of the first women medical school students in Britain and Europe. Emile and Rose most likely met while in medical school in Paris, although for very different reasons, neither graduated. It was previously suggested that Rose left medical school after only a few years, although we present new evidence that that she was still a medical student four years later when she would have been near completion. Regardless, Rose moved back to England prior to taking her qualifying exams, where we found she lived at a girl's boarding school where one of her sisters was head mistress. In the following year, Emile travelled to London where he and Rose were married in a quiet civil ceremony. Soon after the wedding, Emile returned to Paris where he began working as an assistant to Louis Pasteur. In a tragic twist of fate, Rose died a year later in Madeira, which we have now noted was within days of when Emile performed his breakthrough experiments that led to the creation of vaccines in the laboratory.

IR does not dramatically alter spindle geometry of irradiated mitotic cells. Examples of monopolar (A) and multipolar (B) Spindles of U251 cells stained for kinetochores (green), DNA (blue), and microtubules (red). Scale bar, 5-μm. Graphs represent the percentage of monopolar (A) and multipolar (B) spindles as a function of IR dose. Circles represent mean {plus minus} s.e.m, n = 150 cells, 3 experiments.

Control and irradiated (12 Gy) RPE1 cells stained for Aurora-B (A), p-Hec1 (C), p-Histone H3 (E), and p-Cenp-A (G) in green, and DNA (blue). Centromeres (in red) were stained using anti-centromere antibody (CREST). Insets in A show centromere and Aurora B staining at higher magnifications. Relative fluorescence intensity of p-Hec1 (B), p-Histone H3 (D), and p-Cenp-A (F) compared to centromere staining. Bars represent mean {plus minus} SD, n = 10 cells, >300 kinetochores.

IR does not significantly perturb the spindle assembly checkpoint (SAC). A, Experimental schema of assessing the SAC after IR. Noc, Nocodazole. Bar graph depicting mitotic indices as a function of IR dose of Nocodazole-arrested cells 1 hour after irradiation; ns, p>0.2, χ2-test, n=187-523 cells. B, Experimental schema of assessing the SAC after Doxorubicin exposure. Noc, Nocodazole; Doxo, Doxorubicin. Graph depicting the number of mitotic cells after mitotic shakeoff in the absence of nocodazole (control), or in the presence of nocodazole only (Noc), or Nocodazole after a 1-hour exposure to Doxorubicin (Noc + Doxo).

A, Immunoblots of control RPE1 cells and cells depleted for Chk2 using Chk2 siRNA stained using anti-Chk2 antibody and anti-actin antibody as a loading control. B, Chromosome mis-segregation in control RPE1 cells and cells depleted for Chk2 using Chk2 siRNA in the presence or absence of Doxorubicin. Bars represent mean, n = 150 cells, 3 experiments, **, p<0.01. C, Chromosome mis-segregation in AT22IJE-T fibroblasts and AT22IJE-T fibroblasts expressing FLAG-ATM in the presence or absence of either Chloroquine (31μg ml-1) or Doxorubicin (0.5 μM). Bars represent mean, n = 150 cells, 3 experiments, **, p<0.01.

IR does not significantly affect sister chromatid cohesion. A, Experimental schema of assessing sister chromatid cohesion after IR exposure. Noc, Nocodazole. Bar graphs show percentage of mitotic spreads containing chromosomes with intact sister-chromatid cohesion or uncohesed chromosomes as a function of IR dose. n>300 mitotic spreads, p>0.14, χ2-test for all cell lines B, Experimental schema of assessing sister chromatid cohesion after Doxorubicin exposure. Noc, Nocodazole; Doxo, Doxorubicin. Graphs depicting the percentage of mitotic spreads with intact sister chromatid cohesion over time. n>300 spreads, 3 experiments. C, Examples of mitotic spreads from control (left) and irradiated (middle) cells and spreads containing uncohesed chromosomes (right). Scale bar, 25-μm.

A, Example of an anaphase spindle with lagging chromosomes and the number of lagging chromosomes per anaphase spindle as a function of IR dose. B, Example of a multipolar anaphase spindle and the percentage of anaphase spindles with acentric chromatin as a function of IR dose. C, Example of an anaphase spindle with acentric chromatin fragments and the number of multipolar mitoses as a function of IR dose. Circles represent mean {plus minus} s.e.m, n = 150 cells, 3 experiments. Representative mitotic spindles are stained for Hec1 to denote kinetochores (green), DNA (blue), and microtubules (red). Scale bar 5-μm.

Supplementary Figure Legends from DNA-Damage Response during Mitosis Induces Whole-Chromosome Missegregation

Chromosome instability (CIN) is an early step in carcinogenesis that promotes tumor cell progression and resistance to therapy. Using plasmids integrated adjacent to telomeres, we have previously demonstrated that the sensitivity of subtelomeric regions to DNA double-strand breaks (DSBs) contributes to telomere loss and CIN in cancer. A high-throughput screen was created to identify compounds that affect telomere loss due to subtelomeric DSBs introduced by I-SceI endonuclease, as detected by cells expressing green fluorescent protein (GFP). A screen of a library of 1832 biologically-active compounds identified a variety of compounds that increase or decrease the number of GFP-positive cells following activation of I-SceI. A curated screen done in triplicate at various concentrations found that inhibition of classical nonhomologous end joining (C-NHEJ) increased DSB-induced telomere loss, demonstrating that C-NHEJ is functional in subtelomeric regions. Compounds that decreased DSB-induced telomere loss included inhibitors of mTOR, p38 and tankyrase, consistent with our earlier hypothesis that the sensitivity of subtelomeric regions to DSBs is a result of inappropriate resection during repair. Although this assay was also designed to identify compounds that selectively target cells experiencing telomere loss and/or chromosome instability, no compounds of this type were identified in the current screen.

Telomeres, repetitive nucleoprotein complexes that protect chromosomal termini and prevent them from activating inappropriate DNA damage responses (DDRs), shorten with cell division and thus with aging. Here, we characterized the human cellular response to targeted telomeric double-strand breaks (DSBs) in telomerase-positive and telomerase-independent alternative lengthening of telomere (ALT) cells, specifically in G1 phase. Telomeric DSBs in human G1 cells elicited early signatures of a DDR; however, localization of 53BP1, an important regulator of resection at broken ends, was not observed at telomeric break sites. Consistent with this finding and previously reported repression of classical non-homologous end-joining (c-NHEJ) at telomeres, evidence for c-NHEJ was also lacking. Likewise, no evidence of homologous recombination (HR)-dependent repair of telomeric DSBs in G1 was observed. Rather, and supportive of rapid truncation events, telomeric DSBs in G1 human cells facilitated formation of extensive tracks of resected 5′ C-rich telomeric single-stranded (ss)DNA, a previously proposed marker of the recombination-dependent ALT pathway. Indeed, induction of telomeric DSBs in human ALT cells resulted in significant increases in 5′ C-rich (ss)telomeric DNA in G1, which rather than RPA, was bound by the complementary telomeric RNA, TERRA, presumably to protect these exposed ends so that they persist into S/G2 for telomerase-mediated or HR-dependent elongation, while also circumventing conventional repair pathways. Results demonstrate the remarkable adaptability of telomeres, and thus they have important implications for persistent telomeric DNA damage in normal human G1/G0 cells (e.g., lymphocytes), as well as for therapeutically relevant targets to improve treatment of ALT-positive tumors.

The impact of TGFβ signaling on DNA repair competency is observed in a pan-cancer analysis of survival after treatments that cause DNA damage.

DNA polymerase theta (POLQ)-mediated end joining (TMEJ) is a distinct pathway for mediating DNA double-strand break (DSB) repair. TMEJ is required for the viability of BRCA-mutated cancer cells. It is crucial to identify tumors that rely on POLQ activity for DSB repair, because such tumors are defective in other DSB repair pathways and have predicted sensitivity to POLQ inhibition and to cancer therapies that produce DSBs. We define here the POLQ-associated mutation signatures in human cancers, characterized by short insertions and deletions in a specific range of microhomologies. By analyzing 82 COSMIC (Catalogue of Somatic Mutations in Cancer) signatures, we found that BRCA- mutated cancers with a higher level of POLQ expression have a greatly enhanced representation of the small insertion and deletion signature 6, as well as single base substitution signature 3. Using human cancer cells with disruptions ofPOLQ, we further show that TMEJ dominates end joining of two separated DSBs (distal EJ). Templated insertions with microhomology are enriched in POLQ-dependent distal EJ. The use of this signature analysis will aid in identifying tumors relying on POLQ activity.

Repair of DNA damage protects genomic integrity, which is key to tissue functional integrity. In cancer, the type and fidelity of DNA damage response is the fundamental basis for clinical response to cytotoxic therapy. Here we consider the contribution of transforming growth factor-beta (TGFβ), a ubiquitous, pleotropic cytokine that is abundant in the tumor microenvironment, to therapeutic response. The action of TGFβ is best illustrated in head and neck squamous cell carcinoma (HNSCC). Survival of HNSCC patients with human papilloma virus (HPV) positive cancer is more than double compared to those with HPV-negative HNSCC. Notably, HPV infection profoundly impairs TGFβ signaling. HPV blockade of TGFβ signaling, or pharmaceutical TGFβ inhibition that phenocopies HPV infection, shifts cancer cells from error-free homologous-recombination DNA double-strand-break (DSB) repair to error-prone alternative end-joining (altEJ). Cells using altEJ are more sensitive to standard of care radiotherapy and cisplatin, and are sensitized to PARP inhibitors. Hence, HPV-positive HNSCC is an experiment of nature that provides a strong rationale for the use of TGFβ inhibitors for optimal therapeutic combinations that improve patient outcome.

Telomeres are repetitive nucleoprotein complexes that protect the ends of linear chromosomes and prevent their detection as double strand breaks (DSBs), thereby averting activation of a DNA damage response (DDR). While these functions are clearly essential for maintaining genome integrity, it is intriguing to consider how DSBs within telomeres themselves are handled. In cycling cells, telomeric DSBs can be repaired by homologous recombination (HR) and alternative nonhomologous end-joining (ALT NHEJ). Localization of 53BP1, an important regulator of resection at broken ends, to damaged telomeres has also only been observed in replicating cells. Here, we characterized the cellular response to enzymatically induced telomeric DSBs, specifically in non-replicating G1 normal human fibroblasts and cancer cells. Telomeric DSBs in G1 human cells elicited early signatures of a DDR in that gamma (γ)-H2AX and MDC1 were recruited to broken telomeres. Consistent with previous reports, 53BP1 was not observed at telomeric break sites in G1. Furthermore, evidence of classical NHEJ (cNHEJ), the primary pathway of DSB repair in G1 mammalian cells, was lacking at broken telomeres. Likewise, no evidence of classical HR-dependent repair of telomeric DSBs in G1 was observed, as neither RAD51, RAD52, nor repair associated DNA synthesis were detected. Rather, and consistent with rapid truncation events and overall telomere shortening, telomeric DSBs in G1 human cells facilitated formation of extensive tracks of RPA coated 5’ C-rich telomeric single-stranded (ss)DNA, an observation also supported by minimal dependence on conventional end-processing exonucleases MRE11, EXO1, or Apollo. Thus, telomeric DSBs in G1 human cells initiate an abbreviated DDR that results in extensive resection in the absence of 53BP1, facilitating formation of 5’ C-rich overhangs, a previously proposed marker of the recombination dependent, alternative lengthening of telomeres (ALT) pathway, which presumably persist until recombination-mediated elongation and restoration is possible.

Abstract Purpose: Following cytotoxic therapy, 70% of patients with human papillomavirus (HPV)-positive oropharyngeal head and neck squamous cell carcinoma (HNSCC) are alive at 5 years compared with 30% of those with similar HPV-negative cancer. Loss of TGFβ signaling is a poorly studied consequence of HPV that could contribute to patient outcome by compromising DNA repair. Experimental Design: Human HNSCC cell lines (n = 9), patient-derived xenografts (n = 9), tissue microarray (n = 194), TCGA expression data (n = 279), and primary tumor specimens (n = 10) were used to define the relationship between TGFβ competency, response to DNA damage, and type of DNA repair. Results: Analysis of HNSCC specimens in situ and in vitro showed that HPV associated with loss of TGFβ signaling that increased response to radiation or cisplatin. TGFβ suppressed miR-182, which inhibited both BRCA1, necessary for homologous recombination repair (HRR), and FOXO3, required for ATM kinase activity. TGFβ signaling blockade by either HPV or inhibitors released miR182 control, compromised HRR and increased response to PARP inhibition. Antagonizing miR-182 rescued the HRR deficit in HPV-positive cells. Loss of TGFβ signaling unexpectedly increased repair by error prone, alternative end-joining (alt-EJ). Conclusions: HPV-positive HNSCC cells are unresponsive to TGFβ. Abrogated TGFβ signaling compromises repair by HRR and increases reliance on alt-EJ, which provides a mechanistic basis for sensitivity to PARP inhibitors. The effect of HPV in HNSCC provides critical validation of TGFβ’s role in DNA repair proficiency and further raises the translational potential of TGFβ inhibitors in cancer therapy.

Abstract Although human papilloma virus (HPV) status is the most informative prognostic marker in head and neck squamous cell carcinoma (HNSCC), the mechanisms that provide the survival advantage are poorly understood. Here we demonstrate that defects in transforming growth factor β (TGFβ) signaling confer a profound vulnerability in DNA repair for HPV+ HNSCC. We interrogated HNSCC profiled by The Cancer Genome Atlas (TCGA) with a 77 gene chronic TGFβ signature, defined as those genes that were significantly and reciprocally altered in MCF10A cultured for 7 days with TGFβ or a small molecule inhibitor of TGFβ type 1 receptor kinase (TBRIi). The 50 TGFβ-upregulated genes and 27 TGFβ-downregulated genes were negatively correlated (P<0.0001; R2=-0.68) in single-sample gene set enrichment analysis confirmed that the signature is informative in HNSCC. Unsupervised clustering of 294 HNSCC TCGA patient specimens using the TGFβ signature showed that HPV+ cancers were clustered by loss of TGFβ regulation. Patients whose cancers exhibit this profile had significantly better overall survival (P=0.002), independent of HPV status. We previously found that TGFβ signaling suppresses miR-182, which is a reported inhibitor of homologous recombination (HR). HPV+ cell lines increased expression of miR-182 compared to HPV- cell lines. Thus we evaluated radiation induced RAD51 foci formation, a biomarker of HR, in viable explants of patient-derived xenografts (PDX) treated with or without TBRIi. HPV+ PDX showed significantly fewer RAD51 foci than HPV- samples (P<0.005). HPV- PDX specimens treated with TBRIi also reduced RAD51 foci formation. We then established an HPV- cell line containing reporters to measure the frequency of DNA double strand break (DSB) repair with HR or alternative end-joining (Alt-EJ) following DSB induction with I-SceI endonuclease. TBRIi decreased HR events. In contrast, Alt-EJ was significantly increased (P<0.05). Poly (ADP-ribose) polymerase 1 (PARP1) is required for DSB repair with Alt-EJ. TCGA data shows dramatically increased expression of PARP1 in HPV+ HNSCC (P<0.0001). Hence, we speculated that loss of TGFβ signaling increase reliance on PARP1 mediated Alt-EJ to cope with DSB. Consistent with this, HPV+ cell lines were more sensitive to olaparib; while olaparib in combination with TGFβ inhibitor significantly increased cell death in HPV- cell lines by flow cytometry analysis of annexin V positive cells, but not in HPV+ cell lines. Notably, antagonizing miR-182 in HPV+ cells significantly reduced olaparib sensitivity (P<0.05). Together these data indicate HPV suppression of TGFβ signaling in turn shifts DNA repair from error-free HR to hazardous Alt-EJ through upregulated miR-182. Importantly, TGFβ inhibitors can recapitulate this DNA repair deficiency in hard to treat HPV- HNSCC. Citation Format: Qi Liu, Lin Ma, Luis Palomero, Miquel Àngel Pujana, Trevor Jones, Patrick Ha, John Murnane, Mary Helen Barcellos-Hoff. Status of TGFbeta signaling determines PARP inhibitor sensitivity in head and neck 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 2812.

In mammals, DNA double-strand breaks (DSBs) are primarily repaired by classical non-homologous end joining (C-NHEJ), although homologous recombination repair and alternative NHEJ (A-NHEJ), which involve DSB processing, can also occur. These pathways are tightly regulated to maintain chromosome integrity. The ends of chromosomes, called telomeres, contain telomeric DNA that forms a cap structure in cooperation with telomeric proteins to prevent the activation of the DNA damage response and chromosome fusion at chromosome termini. Telomeres and subtelomeric regions are poor substrates for DNA replication; therefore, regions near telomeres are prone to replication fork stalling and chromosome breakage. Moreover, DSBs near telomeres are poorly repaired. As a result, when DSBs occur near telomeres in normal cells, the cells stop proliferating, while in cancer cells, subtelomeric DSBs induce rearrangements due to the absence of cell cycle checkpoints. The sensitivity of subtelomeric regions to DSBs is due to the improper regulation of processing, because although C-NHEJ is functional at subtelomeric DSBs, excessive processing results in an increased frequency of large deletions and chromosome rearrangements involving A-NHEJ.

Abstract Transforming growth factor β (TGFβ) is a well-documented tumor suppressor; a poorly studied aspect of TGFβ biology is its control of genomic stability. Our prior work showed that TGFβ compromises ATM (ataxia telangiectasia mutated) kinase activity, which mediates recognition and repair of DNA damage. Consequently, inhibiting TGFβ following ionizing radiation increases clonogenic death in vitro and tumor control in vivo in breast, brain, and lung cancer preclinical models. As yet unknown is how TGFβ controls ATM kinase. We recently determined that TGFβ regulates mammary lineage commitment by post-transcriptional control of BRCA1 mRNA and protein by its suppression of miR-182 (Martinez-Ruiz et al., Science Signaling, in press). miR-182-mediated downregulation of BRCA1 impacts DNA repair and sensitivity to PARP inhibitors in cancer cell lines (10.1016/j.molcel.2010.12.005) Here we used two cell culture models to investigate the effect of TGFβ regulation of BRCA1 and miR-182 on DNA damage response. Spontaneously immortalized Tgfb1 heterozygote and wild type fibroblasts from primary mammary epithelial preparations were synchronized in S-phase and exposed to UV radiation before fixation to detect unrepaired DNA damage by quantitative image analysis of 53BP1 foci. Consistent with compromised BRCA1 function, more than twice as many cells with 53BP1 foci were evident in Tgfb1 heterozygote fibroblasts compared to wild type cells (p<0.01). The second model consisted of non-malignant human breast epithelial cell line, MCF10A, stably transduced with a scrambled or miR-182 antagomir, grown under serum free conditions and treated with a small molecule inhibitor of TGFβ type I receptor kinase, LY364937. FOXO3 is a target of miR-182 and thus also regulated by TGFβ. and FOXO3 association with ATM is required for its kinase activity (10.1038/ncb1709). As reported, FOXO3 and ATM co-immunoprecipitated, but was eliminated by TGFβ inhibition and this effect was lost in MCF10A cells expressing a miR-182 antagomir. The impaired DNA repair phenotype induced by TGFβ inhibition can be fully rescued by antagonizing miR-182. These studies support TGFβ as stringent regulator of the DNA damage response via suppression miR-182, which directly targets BRCA1 and indirectly affects ATM activity via FOXO3. We predict that cancers that have lost TGFβ signaling capacity will be genomically unstable due to defective DNA damage control, which is consistent with our studies showing that TGFβ inhibition sensitize cancers to radiation therapy. Citation Format: Qi Liu, Haydeliz Martinez-Ruiz, John Murnane, Simon N. Powell, Mary Helen Barcellos-Hoff. TGFβ controls the DNA damage response via miR-182 regulation of BRCA1 and ATM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 831. doi:10.1158/1538-7445.AM2017-831

Telomeres are nucleoprotein structures that are required to protect chromosome ends. Dysfunctional telomeres are recognized as DNA double-strand breaks (DSBs), and elicit the activation of a DNA damage response (DDR). We have previously reported that DSBs near telomeres are poorly repaired, resulting in a high frequency of large deletions and gross chromosome rearrangements (GCRs). Our previous genetic studies have demonstrated that this sensitivity of telomeric regions to DSBs is a result of excessive processing. In the current study, we have further investigated the sensitivity of telomeric regions to DSBs through the analysis of repair proteins associated with DSBs at interstitial and telomeric sites. Following the inducible expression of I-SceI endonuclease, chromatin immunoprecipitation (ChIP) and real-time quantitative PCR were used to compare the recruitment of repair proteins at I-SceI-induced DSBs at interstitial and subtelomeric sites. We observed that proteins that are specifically associated with processing of DSBs during homologous recombination repair, RAD51, BRCA1, and CtIP, are present at a much greater abundance at subtelomeric DSBs. In contrast, Ku70, which is specifically involved in classical nonhomologous end joining, showed no difference at interstitial and subtelomeric DSBs. Importantly, ATM was lower in abundance at subtelomeric DSBs, while ATR was in greater abundance at subtelomeric DSBs, consistent with the accumulation of processed DSBs near telomeres, since processing is accompanied by a transition from ATM to ATR binding. Combined, our results suggest that excessive processing is responsible for the increased frequency of large deletions and GCRs at DSBs near telomeres.