2011 — 2015 |
Bakkenist, Christopher J |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Regulation of Dna Replication Fork Progression by Atm Kinase Activity @ University of Pittsburgh At Pittsburgh
DESCRIPTION (provided by applicant): The long-term goal of this proposal is to understand ATM and ATR kinase signaling at DNA replication forks. While ATM kinase is activated by DNA double strand breaks, ATR kinase is induced by single-stranded (ssDNA) gaps. However, crosstalk exists between the pathways and ATM and ATR phosphorylate an overlapping set of substrates. To identify indispensable ATM kinase signaling we used the ATM kinase inhibitors KU55933 and KU60019 to transiently inhibit ATM kinase activity in cells. Using this innovative approach we showed that the consequences of acute ATM kinase inhibition and ATM protein disruption are distinct. Here we show that acute ATM kinase inhibition arrests DNA synthesis. This is surprising since irradiated cells that express no ATM protein do not arrest DNA synthesis due to a defect in the inhibition of late origin firing. The contribution of chain elongation arrest to the intra-S-phase checkpoint has been difficult to establish since lesions that induce the checkpoint also directly arrest replication forks. While recent evidence indicates that ATR signaling can arrest chain elongation, the role of ATM has not been addressed. We hypothesize that ATM kinase activity promotes the steady progression of replication forks and attenuates ATR kinase activity. We propose that acute ATM kinase inhibition impedes the repair of damaged replication forks causing an accumulation of ssDNA gaps that induce ATR kinase signaling and the intra-S-phase checkpoint. This challenges the paradigm that ATM kinase disruption disables the intra-S-phase checkpoint. Here we will combine the use of KU55933 and KU60019 as sharp tools to inhibit ATM kinase signaling with single DNA fiber-based technology that allows the visualization of multiple origins and individual replication forks emerging from those origins. We will undertake the first investigation of origin density and replication fork velocity in cells following acute ATM kinase inhibition and ATM protein disruption. PUBLIC HEALTH RELEVANCE: ATM kinase inhibitors are being developed as clinical radiosensitizing agents. We have shown that ATM kinase has significant roles during the DNA replication process. We propose that the great promise of ATM kinase inhibitors lies in their potential as stand alone agents for the effective treatment of 10,000s of cancers that experience replication stress as a consequence of acquired mutations every year.
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2017 — 2021 |
Bakkenist, Christopher J. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Dna Damage Signaling to Immune Checkpoints @ University of Pittsburgh At Pittsburgh
Lung cancer is diagnosed late, has a 5 year survival of only 15%, and kills more people than colorectal, breast and prostate cancer combined. Less than 30% of lung cancers are resected and the majority of patients are treated with cisplatin and ionizing radiation (IR). We show that inhibition of DNA damage signaling by ATR kinase during treatment with cisplatin and IR is well-tolerated and leads to durable responses in mouse xenograft and genetic models of lung cancer. Quite unexpectedly, we show that in addition to potentiating DNA damage, two clinical ATR kinase inhibitors (ATRi?s), with unrelated structures, block expression of the immune checkpoint protein PD-L1 and increase presentation of MHC class I antigens in lung cancer cells after IR. Our finding that crosstalk exists between DNA damage signaling and immune checkpoints has not been described previously and is the focus of this proposal. Immune-inhibitory pathways, termed immune checkpoints, are coopted by tumor cells to evade cytotoxic immune cells. PD-1 is expressed on cytotoxic T cells and its ligand PD-L1 is upregulated in lung cancers. PD-L1 binding by PD-1 prevents the activation of cytotoxic T cells. Immune checkpoint blockade using anti-PD-L1 and anti-PD-1 antibodies restores anti-tumor immune responses and is emerging as an exciting lung cancer therapy. We propose that ATRi?s inhibit DNA repair and cell cycle checkpoints potentiating the DNA damage induced by cisplatin and IR while concurrently inhibiting PD-L1 expression and restoring anti-tumor immune responses. Our objective in this proposal is to define the mechanisms that connect DNA damage signaling and immune checkpoints. This objective will be accomplished by the following Specific Aims. Aim 1: To determine how ATRi?s inhibit PD-L1 expression in lung cancer cells after IR. This aim will define the contribution of ATR, ATM, IRF-1, NF-?B and p53 to PD-L1 expression after IR. Aim 2: To determine how ATRi?s increase MHC class I expression on lung cancer cells after IR. This aim will identify ATR, ATM, and p53 signaling that inhibits protein synthesis and MHC class I presentation after IR. Aim 3: To identify ATRi-induced PDL-1/PD-1 immune checkpoint blockade in lung cancer after IR. This aim will identify ATRi-induced immune checkpoint blockade in lung cancer after IR. The outcomes of these Aims will identify mechanisms that connect DNA damage signaling to immune self- tolerance. This will define a novel therapeutic opportunity to use ATRi?s to potentiate the DNA damage induced by cisplatin and IR while concurrently inducing immune checkpoint blockade.
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2019 — 2020 |
Bakkenist, Christopher J. Boone, David |
R25Activity Code Description: For support to develop and/or implement a program as it relates to a category in one or more of the areas of education, information, training, technical assistance, coordination, or evaluation. |
The Upmc Hillman Cancer Center Academy @ University of Pittsburgh At Pittsburgh
PROJECT SUMMARY/ABSTRACT Over the past nine years, the Hillman Cancer Center (HCC) at the University of Pittsburgh Medical Center (UPMC) has trained and mentored 134 high school students from underrepresented minority (URM) groups and economically disadvantaged (DA) backgrounds hailing from over 60 high schools. Our newly proposed Hillman Academy will expand the successes of our current program, which is funded under the expiring Continuing Umbrella of Research Experiences to the HCC and was recognized in 2014 with the Carnegie Science Center Leadership in STEM (Science, Technology, Engineering, and Mathematics) Education Award. Our past participants performed research over a wide range of cancer topics at six research sites across the campus under the tutelage of 106 faculty members who supported our students as hosts and mentors. Our alumni have won numerous scholarships and awards, coauthored manuscripts, presented at national scientific conferences, and continued research both with us and at the top institutions that they now attend. Ninety-three percent of our alumni with known and declared undergraduate majors matriculated into science- and healthcare-related areas of study. We aim to continue these successes with an expanded program to reach even more URM/DA students?24 per year?to increase the diversity and preparedness of the cancer workforce through a multi-modal approach that includes immersive research experiences, didactic training with tailored curricula, and professional and academic development. We will also lead a number of outreach activities in our region to raise awareness of cancer, cancer disparities, and cancer research through our collaborations with long-standing community partners, such as the Fund for the Advancement of Minorities through Education, MPowerhouse, Inc., and the Homeless Children?s Education Fund. These efforts will grow out of the Hillman Academy to engage URM/DA students at earlier stages, as well as, their schools and other community members. Together, the Hillman Academy will engage, train, and provide paths for URM/DA youth to advance cancer research and care. Our leadership, infrastructure, mentors, and experience of running a holistic research training program position us for lasting success in this endeavor. We will build on our previous accomplishments of promoting the pursuit of STEM education and careers for our students and continue to address cancer disparities by equipping URM/DA youth to be agents of change that impact their communities.
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2020 — 2021 |
Bakkenist, Christopher J. |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Cancer Research Career Enhancement and Related Activities @ University of Pittsburgh At Pittsburgh
Abstract: Career Enhancement and Related Activities A major strategic initiative for UPMC Hillman Cancer Center (HCC) is to develop a well-trained, diverse biomedical workforce through research education, training, and career-development activities that promote the understanding of the biology and treatment of cancer. To this end, the HCC Committee for Excellence in Cancer Education and Training (CECET) meets 4 times a year to review the breadth, depth, accessibility, and effectiveness of available education and training opportunities and to develop new programs as needed. CECET overseas an HCC investment of ~$1.4M/year in education and training. Our work is organized through three main specific aims designed to provide career enhancement opportunities across the biomedical training pipeline. The first aim is to educate and inspire high school and undergraduate students about careers in cancer research with a focus on serving underrepresented populations. The Hillman Academy has provided an 8-week summer internship introducing careers in cancer research to 433 high school students, including 175 (40%) underrepresented minority and disadvantaged (URM/DA) in our catchment area (CA). The Academy is supported by the Doris Duke Charitable Foundation, CURE and the Jack Kent Foundation, and the award of a new R25CA236620 allows an immediate 70% increase in the number of URM/DA students. This new R25CA236620 also provides support to improve the science literacy of our surrounding community by developing 1-day workshops that include hands-on activities, tours of facilities, and career seminars on cancer research. Our second aim is to encourage medical and doctoral students as well as clinical and postdoctoral fellows to pursue careers in cancer research. We have used HCC philanthropy to recruit the first class of Hillman Medical Student and Postdoctoral Fellows for Innovative Cancer Research. We have increased the number of NCI T32 training grants from 4 to 6 to support the cancer research focus of all CCSG programs. Our commitment to graduate student researcher (GSR) education is evidenced by our support of more GSRs than any other Pitt SOM Institute or Department. At the request of the newly formed HCC postdoctoral association, we have established a new grant writing class that tailors K99 R00 applications to the NCI. This initiative builds on previous F30, F31, F99 K00 and K00 R01 successes. We will extend our Hillman Fellows for Innovative Cancer Research program into a new ?Bridge to Faculty? positions that will guarantee faculty positions when key metrics are attained. Our third aim is to enhance the professional growth of faculty through an extensive, multi-faceted faculty mentorship program that eliminates barriers to innovative cancer research and strengthens R01 applications to the NCI. We will also increase efforts across our network to ensure equal access of our health care providers to exceptional cancer education and training programs, such as Accountability for Cancer Care through Undoing Racism and Equity and AP-POWER (R25CA148050).
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2020 — 2021 |
Bakkenist, Christopher J. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Dna Damage Signaling to Dormant Origins of Replication @ University of Pittsburgh At Pittsburgh
Higher eukaryotes evolved with mechanisms that initiate DNA replication at multiple origins on multiple chromosomes. Activation of the replicative helicase at a single origin in each of ~50,000 replicons is sufficient to replicate the human genome in the absence of stress. These ~50,000 origins are selected from a five- to tenfold excess of licensed origins. Activation of additional replicative helicases at origins that would otherwise be passively replicated is observed after stress. This plasticity in origin use is a simple mechanism to recover DNA replication between stalled and collapsed replication forks. The mechanism(s) that limits origin firing to one per replicon in the absence of stress is not known. We recently reported that the DNA damage signaling kinases ATR and Chk1 inhibit activation of the replicative helicase in the absence of stress. In preliminary studies, we show that Chk1 kinase activity is strictly associated with ATR kinase-dependent phosphorylations on Chk1 and that these have an astonishingly short half-life in cells. We propose that this is a highly innovative mechanism that localizes Chk1 kinase activity to the immediate vicinity of ATR at active replicative helicases. We also show that Rif1, which has been implicated in the regulation of replication timing previously, is phosphorylated and that phosphorylated Rif1 binds protein phosphatase 1? (PP1?). Based upon these findings, we hypothesize that Chk1 kinase activity generates a ring of Rif1-PP1? around active replicative helicases and that this limits Cdc7 kinase-dependent origin firing across a replicon. In Aim 1 we will investigate a new mechanistic paradigm for localizing DNA damage signaling to a small volume of the nucleus in the absence of stress. In Aim 2 we will investigate the molecular mechanism that limits activation of the replicative helicase across a replicon in the absence of stress. In Aim 3 we will investigate the impact of ATR kinase inhibition and conformal radiation on immune responses in tumor bearing mice. These studies are highly impactful as they will identify a fundamental mechanism that determines inter-origin distance, genome stability and the rate of cell division in higher eukaryotes. Since this mechanism may be attenuated in T cells, these studies will provide fundamental insights into adaptive immune responses. Our studies may have an immediate impact as the ATR and Chk1 kinase inhibitors used here are in clinical trials.
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