Jean-Bernard Lazaro

DNA-Dependent Protein Kinase (DNA-PK)–Dependent Cisplatin-Induced Loss of Nucleolar Facilitator of Chromatin Transcription (FACT) and Regulation of Cisplatin Sensitivity by DNA-PK and FACT

Both the Ku subunit of the DNA-dependent protein kinase (DNA-PK) and the facilitator of chromatin transcription (FACT) complex reportedly bind cisplatin-DNA adducts. For this study, we developed an immunocytochemical assay based on detergent extraction allowing unveiling nucleolar subpopulations of proteins present in both the nucleoplasm and the nucleolus. Immunofluorescence analysis in various human cancer cell lines and immunoblotting of isolated nucleoli show that DNA-PK catalytic subunit (DNA-PKcs), Ku86, the Werner syndrome protein (WRN), and the structure-specific recognition protein 1 (SSRP1) subunit of FACT colocalize in the nucleolus and exit the nucleolus after cisplatin treatment. Nucleolar localization of Ku is also lost after γ or UV irradiation and exposure to DNA-damaging drugs, such as actinomycin D, mitomycin C, hydroxyurea, and doxorubicin. Ku86 and WRN leave the nucleolus after exposure to low (>1 μg/mL) doses of cisplatin. In contrast, the SSRP1 association with the nucleolus was disrupted only by high (50-100 μg/mL) doses of cisplatin. Both cisplatin-induced loss of nucleolar SSRP1 and DNA-PK activation are suppressed by pretreatment of the cells with wortmannin or the DNA-PK inhibitor NU7026 but not by the phosphatidylinositol 3-kinase inhibitor LY294002. In the same conditions, kinase inhibitors did not alter the exit of DNA-PKcs and WRN, suggesting that different mechanisms regulate the exit of DNA-PK/WRN and FACT from the nucleolus. Furthermore, RNA silencing of DNA-PKcs blocked the cisplatin-induced exit of nucleolar SSRP1. Finally, silencing of DNA-PKcs or SSRP1 by short hairpin RNA significantly increased the sensitivity of cancer cells to cisplatin.(Mol Cancer Res 2009;7(4):581–91)

Online Nanoflow RP−RP-MS Reveals Dynamics of Multicomponent Ku Complex in Response to DNA Damage

Tandem affinity purification (TAP) coupled with mass spectrometry has become the technique of choice for characterization of multicomponent protein complexes. While current TAP protocols routinely provide high yield and specificity for proteins expressed under physiologically relevant conditions, analytical figures of merit required for efficient and in-depth LC-MS analysis remain unresolved. Here we implement a multidimensional chromatography platform, based on two stages of reversed-phase (RP) separation operated at high and low pH, respectively. We compare performance metrics for RP-RP and SCX-RP for the analysis of complex peptide mixtures derived from cell lysate, as well as protein complexes purified via TAP. Our data reveal that RP-RP fractionation outperforms SCX-RP primarily due to increased peak capacity in the first dimension separation. We integrate this system with miniaturized LC assemblies to achieve true online fractionation at low (≤5 nL/min) effluent flow rates. Stable isotope labeling is used to monitor the dynamics of the multicomponent Ku protein complex in response to DNA damage induced by γ radiation.

Fast kinase domain-containing protein 3 is a mitochondrial protein essential for cellular respiration

Fas-activated serine/threonine phosphoprotein (FAST) is the founding member of the FAST kinase domain-containing protein (FASTKD) family that includes FASTKD1-5. FAST is a sensor of mitochondrial stress that modulates protein translation to promote the survival of cells exposed to adverse conditions. Mutations in FASTKD2 have been linked to a mitochondrial encephalomyopathy that is associated with reduced cytochrome c oxidase activity, an essential component of the mitochondrial electron transport chain. We have confirmed the mitochondrial localization of FASTKD2 and shown that all FASTKD family members are found in mitochondria. Although human and mouse FASTKD1-5 genes are expressed ubiquitously, some of them are most abundantly expressed in mitochondria-enriched tissues. We have found that RNA interference-mediated knockdown of FASTKD3 severely blunts basal and stress-induced mitochondrial oxygen consumption without disrupting the assembly of respiratory chain complexes. Tandem affinity purification reveals that FASTKD3 interacts with components of mitochondrial respiratory and translation machineries. Our results introduce FASTKD3 as an essential component of mitochondrial respiration that may modulate energy balance in cells exposed to adverse conditions by functionally coupling mitochondrial protein synthesis to respiration.

DNA damage-induced inhibition of rRNA synthesis by DNA-PK and PARP-1

RNA synthesis and DNA replication cease after DNA damage. We studied RNA synthesis using an in situ run-on assay and found ribosomal RNA (rRNA) synthesis was inhibited 24 h after UV light, gamma radiation or DNA cross-linking by cisplatin in human cells. Cisplatin led to accumulation of cells in S phase. Inhibition of the DNA repair proteins DNA-dependent protein kinase (DNA-PK) or poly(ADP-ribose) polymerase 1 (PARP-1) prevented the DNA damage-induced block of rRNA synthesis. However, DNA-PK and PARP-1 inhibition did not prevent the cisplatin-induced arrest of cell cycle in S phase, nor did it induce de novo BrdU incorporation. Loss of DNA-PK function prevented activation of PARP-1 and its recruitment to chromatin in damaged cells, suggesting regulation of PARP-1 by DNA-PK within a pathway of DNA repair. From these results, we propose a sequential activation of DNA-PK and PARP-1 in cells arrested in S phase by DNA damage causes the interruption of rRNA synthesis after DNA damage.

Concordant and opposite roles of DNA-PK and the "facilitator of chromatin transcription" (FACT) in DNA repair, apoptosis and necrosis after cisplatin

BackgroundPlatinum-containing chemotherapy produces specific DNA damage and is used to treat several human solid tumors. Tumors initially sensitive to platinum-based drugs frequently become resistant. Inhibition of DNA repair is a potential strategy to enhance cisplatin effectiveness. After cisplatin treatment, a balance between repair and apoptosis determines whether cancer cells proliferate or die. DNA-dependent protein kinase (DNA-PK) binds to DNA double strand breaks (DSBs) through its Ku subunits and initiates non-homologous end joining. Inhibition of DNA-PK sensitizes cancer cells to cisplatin killing. The goal of this study is to elucidate the mechanism underlying the effects of DNA-PK on cisplatin sensitivity.ResultsSilencing the expression of the catalytic subunit of DNA-PK (DNA-PKcs) increased sensitivity to cisplatin and decreased the appearance of γH2AX after cisplatin treatment. We purified DNA-PK by its Ku86 subunit and identified interactors by tandem mass spectrometry before and after cisplatin treatment. The structure specific recognition protein 1 (SSRP1), Spt16 and γH2AX appeared in the Ku86 complex 5 hours after cisplatin treatment. SSRP1 and Spt16 form the facilitator of chromatin transcription (FACT). The cisplatin-induced association of FACT with Ku86 and γH2AX was abrogated by DNase treatment. In living cells, SSRP1 and Ku86 were recruited at sites of DSBs induced by laser beams. Silencing SSRP1 expression increased sensitivity to cisplatin and decreased γH2AX appearance. However, while silencing SSRP1 in cisplatin-treated cells increased both apoptosis and necrosis, DNA-PKcs silencing, in contrast, favored necrosis over apoptosis.ConclusionsDNA-PK and FACT both play roles in DNA repair. Therefore both are putative targets for therapeutic inhibition. Since DNA-PK regulates apoptosis, silencing DNA-PKcs redirects cells treated with cisplatin toward necrosis. Silencing FACT however, allows both apoptosis and necrosis. Targeting DNA repair in cancer patients may have different therapeutic effects depending upon the roles played by factors targeted.

NF-κB inhibition by dimethylaminoparthenolide radiosensitizes non-small-cell lung carcinoma by blocking DNA double-strand break repair

Despite optimal chemotherapy, radiotherapy (RT), and/or surgery, non-small-cell lung carcinoma (NSCLC) remains the leading cause of cancer-related death in the US and worldwide. Thoracic RT, a mainstay in the treatment of locally advanced NSCLC, is often restricted in efficacy by a therapeutic index limited by sensitivity of tissues surrounding the malignancy. Therefore, radiosensitizers that can improve the therapeutic index are a vital unmet need. Inhibition of the NF-κB pathway is a proposed mechanism of radiosensitization. Here we demonstrate that inhibition of the canonical NF-κB pathway by dimethylaminoparthenolide (DMAPT) radiosensitizes NSCLC by blocking DNA double-strand break (DSB) repair. NF-κB inhibition results in significant impairment of both homologous recombination (HR) and non-homologous end joining (NHEJ), as well as reductions in ionizing radiation (IR)-induced DNA repair biomarkers. NF-κB inhibition by DMAPT shows preclinical potential for further investigation as a NSCLC radiosensitizer.

Abstract PR18: Somatic ERCC2 mutations, nucleotide excision repair (NER) function, and cisplatin response in muscle-invasive bladder cancer (MIBC)

ERCC2 is a core member of the nucleotide excision repair (NER) pathway, a highly conserved and remarkably versatile DNA repair pathway responsible for repairing intrastrand DNA adducts created by genotoxic agents such as UV irradiation and platinum-based chemotherapies. Recent large-scale genomic efforts have shown that somatic ERCC2 missense mutations are present in approximately 20% of all primary muscle-invasive bladder cancers (MIBC). We previously showed that ERCC2 mutations are associated with treatment response and overall survival in MIBC patients treated with cisplatin-based chemotherapy. Initial functional studies on a subset of the observed ERCC2 mutations suggest that the mutations confer loss of normal cellular NER capacity. However, sequencing of additional MIBC cohorts has revealed that mutations occur across the ERCC2 gene, and the functional effects of the majority of these mutations remain unknown. In order to understand the functional landscape of ERCC2 mutations in MIBC, we have developed a high-throughput fluorescence-based assay to test the functional consequences of mutations in ERCC2 and other NER genes on cellular NER capacity. We apply this approach to all observed ERCC2 mutations across three published MIBC cohorts and find that the majority of ERCC2 mutations result in complete or near-complete loss of cellular NER. In addition, by correlating our functional results with available clinical data, we find interesting examples of cases in which ERCC2 status and cisplatin response are decoupled, highlighting the importance of using functional data to complement genomic and clinical endpoints in the search for reliable predictive biomarkers. This abstract is also being presented as Poster A29. Citation Format: Kent Mouw, Jean-Bernard Lazaro, Alexis Damish, Elizaveta Reznichenko, Zoe Frazier, David Liu, Jaegil Kim, Paz Polak, Levi Garraway, Gad Getz, Jonathan Rosenberg, Eliezer Van Allen, Alan D9Andrea. Somatic ERCC2 mutations, nucleotide excision repair (NER) function, and cisplatin response in muscle-invasive bladder cancer (MIBC) [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr PR18.

Abstract 2796: Development of a RAD51-based assay for determining homologous recombination proficiency and PARP inhibitor sensitivity

Homologous recombination (HR) repair deficiency confers sensitivity to inhibitors of poly(ADP-ribose) polymerase (PARP). To date, the identification of tumors with impaired HR has relied on genomic features, including mutational signature, LOH-based HRD assays or gene expression analyses defining ‘BRCAness’. These tests analyze history of the tumor rather than providing a functional assessment of HR status at the time of diagnosis. Therefore, development of a functional assay for HR status in tumors is essential to make accurate treatment decisions. Here, we describe a RAD51-based immunohistochemical (IHC) assay that identifies HR status. We first screened commercial anti-RAD51 antibodies and identified a monoclonal antibody that detects RAD51 foci in HR-competent normal fibroblasts and shows no evidence of foci in HR-deficient (BRCA2-/-) VU423 fibroblasts after γ-irradiation. Conditions for detecting RAD51 foci in FFPE samples were identified using HR-deficient and HR-proficient triple-negative breast cancer cell lines. HR-deficient, PARP inhibitor-sensitive cell lines exhibited high levels of nuclear RAD51 and no evidence of foci, whereas HR-proficient, PARP inhibitor-resistant cells had low levels of nuclear RAD51 and foci. This result was confirmed in a BRCA1-mutated, PARP inhibitor-sensitive PDX model, where there was no evidence of foci although RAD51 levels were high. We further evaluated the pattern of RAD51 staining in 13 high-grade serous ovarian cancer (HGSOC) PDX models, for which sensitivity to olaparib had been characterized. The only olaparib-sensitive model demonstrated complete absence of RAD51 staining. Among the other 12 olaparib-resistant models, RAD51 foci were detectable, both before and after irradiation. The presence or absence of RAD51 foci correlated with olaparib sensitivity and not with BRCA mutation status. Therefore, tumors that are HR-deficient and PARP inhibitor-sensitive are characterized by either high RAD51 nuclear staining without foci, or absence of RAD51 staining. To validate these findings, we analyzed RAD51 staining patterns in a cohort of 50 primary HGSOCs from patients subsequently treated with platinum-based chemotherapy. Among these 50 samples, 45 demonstrated either RAD51 nuclear staining without foci or an absence of RAD51 staining. Five samples demonstrated RAD51 staining with foci. The median survivals of these groups were 6.1 and 1.5 years, respectively. In conclusion, we have developed a robust IHC assay for determining the functional HR-status in tumor samples. Further work will be required to determine if the staining patterns observed predict PARP inhibitor sensitivity among primary patient samples. Funded by a Biomarker Supplement to UM1 CA186709, NIH Grant P50 CA168504, SU2C Ovarian Cancer Dream Team and BCRF grant. Citation Format: Bose S. Kochupurakkal, Kalindi Parmar, Jean-Bernard Lazaro, Christine Unitt, Qing Zeng, Hunter Reavis, Chirag Ganesa, Shan Zhou, Joyce Liu, Sangeetha Palakurthi, Kyle Strickland, Brooke Howitt, Panagiotis Konstantinopoulos, Paul Kirschmeier, Joseph Geradts, Ronny Drapkin, Ursula Matulonis, Alan D9Andrea, Geoffrey Shapiro. Development of a RAD51-based assay for determining homologous recombination proficiency and PARP inhibitor sensitivity [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 2796. doi:10.1158/1538-7445.AM2017-2796

Abstract 2729: Estrogen receptor-negative breast cancer cell lines exhibit hypersensitivity to the CHK1 inhibitor LY2606368

Checkpoint kinase 1 (CHK1) inhibitors are currently under investigation as chemopotentiating agents due to the role of CHK1 in establishing DNA damage checkpoints in the cell cycle. A novel CHK1/CHK2 inhibitor, LY2606368, as a single agent causes replication catastrophe, DNA double strand breaks and apoptosis (King C et al. Mol Cancer Ther. 2015). Accordingly, LY2606368 is currently in clinical development as a single agent and in combination with both cytotoxic and targeted agents. As subsets of breast cancers exhibit genomic instability and DNA repair deficiencies, we assessed the effect of LY2606368 as a single agent on breast cancer cell lines. We characterized the IC509s for growth inhibition by LY2606368 of a large panel of cell lines assembled on the basis of estrogen receptor (ER), progesterone receptor (PR), and HER2 expression status. Triple negative breast cancer (TNBC) is a subtype of breast cancer that is pathologically negative for expression of ER/PR and HER2 protein. As previously reported for other CHK1 inhibitors (Bryant C et al. BMC Cancer. 2014; Albiges L et al. Breast. 2014), TNBC cell lines exhibited high sensitivity to LY2606368. Interestingly, hypersensitivity to LY2606368 was observed in ER-negative cells, regardless of HER2 status. In addition, ER-positive cells were comparatively resistant suggesting that high sensitivity to LY2606368 occurs in the absence of ER and is not restricted to TNBC. No correlation was found between TP53 mutational status and sensitivity to LY2606368. Consistent with the observed hypersensitivity, ER-negative cell lines exposed to LY2606368 exhibited high levels of γH2AX and phospho-Ser1981-ATM demonstrating appearance of DNA double strand breaks. The concomitant appearance of phospho-Ser345-Chk1 marked aborted checkpoint activation by upstream detector/effector kinases (e.g. ATR, ATM). ER-positive cells did not engage in initiation of the DNA damage response or significant checkpoint activation when exposed to comparable doses of LY2606368. Collectively, these results suggest that the CHK1 inhibitor LY2606368 is likely to be more cytotoxic in ER-negative breast cancer types. Homologous recombination and other DNA repair deficiencies can explain the need for CHK1-dependent checkpoint activation during each replication cycle. On the other hand, ER-positive breast cancers might be less sensitive to monotherapy because of the absence of unresolved DNA damaging events that create checkpoint dependency. In summary, triple negative status does not directly determine the sensitivity of breast cancer cell lines to the CHK1 inhibitor LY2606368, although TNBC cells are in general hypersensitive. The absence of ER might be a more promising marker of sensitivity to LY2606368 as a monotherapy in breast cancer. Citation Format: Jean-Bernard Lazaro, Kalindi Parmar, Geoffrey I. Shapiro, Alan D. D’Andrea. Estrogen receptor-negative breast cancer cell lines exhibit hypersensitivity to the CHK1 inhibitor LY2606368. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2729.

Abstract 1644: NF-κB inhibitor DMAPT blocks non-homologous end-joining repair of radiation-induced DSBs in NSCLC

Background: Despite optimal multimodality therapy, non-small cell lung carcinoma (NSCLC) remains the leading cause of cancer-related death in the United States. A common limitation is our inability to provide sufficient radiotherapy (RT) to eradicate tumor due to risk of toxicities in surrounding tissues. There is thus an unmet need for radiosensitizers that can improve the therapeutic index. Dimethylaminoparthenolide (DMAPT), an orally bioavailable small molecule NF-κB inhibitor, inhibits repair of ionizing radiation (IR)-induced DNA double strand breaks (DSBs) and increases control of subcutaneous mouse NSCLC xenografts. While our prior work focused on inhibition of homologous recombination as a mechanism for radiosensitization, we sought to characterize the effect of DMAPT on a second major DSB repair pathway, non-homologous end-joining (NHEJ). Methods: NHEJ was assessed in NSCLC lines using the pEJ reporter and flow cytometry. The NF-κB super repressor (IκBαS32A,S36A) was used as a control. Immunofluorescence and Western blotting were used to assess NHEJ biomarkers including 53BP1, DNA-PKS2056, Ku70/80 and XRCC4. Cell fractionation was performed to assess Ku chromatin binding. Quantitative RT-PCR was performed to assess gene transcription. Ku complexes were purified to identify binding partners. Results: NSCLC cells treated with IR-sensitizing doses of DMAPT (5-15 μM) or the NF-κB super repressor showed significant decreases in NHEJ. DMAPT increased the persistence of 53BP1 foci, indicating a failure to complete NHEJ. Regardless of exogenous DNA damage, there was reduced Ku70 chromatin binding following DMAPT treatment. DMAPT-treated cells produced fewer distinct IR-induced DNA-PKS2056 foci. Further, there was decreased IR-induced XRCC4 chromatin recruitment, suggesting that repair was impaired prior to ligation. There was no change in Ku70/80 transcription following DMAPT and/or IR. However, Western blotting of purified Ku complexes showed that DMAPT treatment decreased Ku association with RNA binding partners including RPL19. We also observed decreased recruitment of DNA-PKcs to the Ku complex, suggesting decreased Ku affinity for DSBs. Conclusions: These results indicate that DMAPT radiosensitizes NSCLC by perturbing the binding affinity of Ku to RNA, DNA and its complex binding partners, thus blocking NHEJ. Further mechanistic investigation and analysis of NHEJ biomarkers in vivo is needed to identify the precise mechanism. Citation Format: Peter V. Deraska, Colin O’Leary, Jean-Bernard Lazaro, Christopher J. Sweeney, Alan D. D’Andrea, David Kozono. NF-κB inhibitor DMAPT blocks non-homologous end-joining repair of radiation-induced DSBs in NSCLC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1644.