Ryan L. Ragland

Combining ATR suppression with oncogenic Ras synergistically increases genomic instability, causing synthetic lethality or tumorigenesis in a dosage-dependent manner

Previous studies indicate that oncogenic stress activates the ATR-Chk1 pathway. Here, we show that ATR-Chk1 pathway engagement is essential for limiting genomic instability following oncogenic Ras transformation. ATR pathway inhibition in combination with oncogenic Ras expression synergistically increased genomic instability, as quantified by chromatid breaks, sister chromatid exchanges, and H2AX phosphorylation. This level of instability was significantly greater than that observed following ATR suppression in untransformed control cells. In addition, consistent with a deficiency in long-term genome maintenance, hypomorphic ATR pathway reduction to 16% of normal levels was synthetic lethal with oncogenic Ras expression in cultured cells. Notably, elevated genomic instability and synthetic lethality following suppression of ATR were not due to accelerated cycling rates in Ras-transformed cells, indicating that these synergistic effects were generated on a per-cell-cycle basis. In contrast to the synthetic lethal effects of hypomorphic ATR suppression, subtle reduction of ATR expression (haploinsufficiency) in combination with endogenous levels of K-ras(G12D) expression elevated the incidence of lung adenocarcinoma, spindle cell sarcoma, and thymic lymphoma in p53 heterozygous mice. K-ras(G12D)-induced tumorigenesis in ATR(+/-)p53(+/-) mice was associated with intrachromosomal deletions and loss of wild-type p53. These findings indicate that synergistic increases in genomic instability following ATR reduction in oncogenic Ras-transformed cells can produce 2 distinct biological outcomes: synthetic lethality upon significant suppression of ATR expression and tumor promotion in the context of ATR haploinsufficiency. These results highlight the importance of the ATR pathway both as a barrier to malignant progression and as a potential target for cancer treatment.

Oncogenic stress sensitizes murine cancers to hypomorphic suppression of ATR

Oncogenic Ras and p53 loss-of-function mutations are common in many advanced sporadic malignancies and together predict a limited responsiveness to conventional chemotherapy. Notably, studies in cultured cells have indicated that each of these genetic alterations creates a selective sensitivity to ataxia telangiectasia and Rad3-related (ATR) pathway inhibition. Here, we describe a genetic system to conditionally reduce ATR expression to 10% of normal levels in adult mice to compare the impact of this suppression on normal tissues and cancers in vivo. Hypomorphic suppression of ATR minimally affected normal bone marrow and intestinal homeostasis, indicating that this level of ATR expression was sufficient for highly proliferative adult tissues. In contrast, hypomorphic ATR reduction potently inhibited the growth of both p53-deficient fibrosarcomas expressing H-rasG12V and acute myeloid leukemias (AMLs) driven by MLL-ENL and N-rasG12D. Notably, DNA damage increased in a greater-than-additive fashion upon combining ATR suppression with oncogenic stress (H-rasG12V, K-rasG12D, or c-Myc overexpression), indicating that this cooperative genome-destabilizing interaction may contribute to tumor selectivity in vivo. This toxic interaction between ATR suppression and oncogenic stress occurred without regard to p53 status. These studies define a level of ATR pathway inhibition in which the growth of malignancies harboring oncogenic mutations can be suppressed with minimal impact on normal tissue homeostasis, highlighting ATR inhibition as a promising therapeutic strategy.

RNF4 and PLK1 are required for replication fork collapse in ATR-deficient cells

The ATR-CHK1 axis stabilizes stalled replication forks and prevents their collapse into DNA double-strand breaks (DSBs). Here, we show that fork collapse in Atr-deleted cells is mediated through the combined effects the sumo targeted E3-ubiquitin ligase RNF4 and activation of the AURKA-PLK1 pathway. As indicated previously, Atr-deleted cells exhibited a decreased ability to restart DNA replication following fork stalling in comparison with control cells. However, suppression of RNF4, AURKA, or PLK1 returned the reinitiation of replication in Atr-deleted cells to near wild-type levels. In RNF4-depleted cells, this rescue directly correlated with the persistence of sumoylation of chromatin-bound factors. Notably, RNF4 repression substantially suppressed the accumulation of DSBs in ATR-deficient cells, and this decrease in breaks was enhanced by concomitant inhibition of PLK1. DSBs resulting from ATR inhibition were also observed to be dependent on the endonuclease scaffold protein SLX4, suggesting that RNF4 and PLK1 either help activate the SLX4 complex or make DNA replication fork structures accessible for subsequent SLX4-dependent cleavage. Thus, replication fork collapse following ATR inhibition is a multistep process that disrupts replisome function and permits cleavage of the replication fork.

Targeting the ATR/CHK1 Axis with PARP Inhibition Results in Tumor Regression in BRCA-Mutant Ovarian Cancer Models

Purpose: PARP inhibition (PARPi) has modest clinical activity in recurrent BRCA-mutant (BRCAMUT) high-grade serous ovarian cancers (HGSOC). We hypothesized that PARPi increases dependence on ATR/CHK1 such that combination PARPi with ATR/CHK1 blockade results in increased cell death and tumor regression. Experimental Design: Effects of PARPi (olaparib), CHK1 inhibition (CHK1i;MK8776), or ATR inhibition (ATRi;AZD6738) alone or in combination on survival, colony formation, cell cycle, genome instability, and apoptosis were evaluated in BRCA1/2MUT HGSOC cells. Tumor growth in vivo was evaluated using a BRCA2MUT patient-derived xenograft (PDX) model. Results: PARPi monotherapy resulted in a decrease in BRCAMUT cell survival, colony formation and suppressed but did not eliminate tumor growth at the maximum tolerated dose (MTD) in a BRCA2MUT PDX. PARPi treatment increased pATR and pCHK1, indicating activation of the ATR–CHK1 fork protection pathway is relied upon for genome stability under PARPi. Indeed, combination of ATRi or CHK1i with PARPi synergistically decreased survival and colony formation compared with single-agent treatments in BRCAMUT cells. Notably, PARPi led to G2 phase accumulation, and the addition of ATRi or CHK1i released cells from G2 causing premature mitotic entry with increased chromosomal aberrations and apoptosis. Moreover, the combinations of PARPi with ATRi or CHK1i were synergistic in causing tumor suppression in a BRCA2MUT PDX with the PARPi–ATRi combination inducing tumor regression and in most cases, complete remission. Conclusions: PARPi causes increased reliance on ATR/CHK1 for genome stability, and combination PARPi with ATR/CHK1i is more effective than PARPi alone in reducing tumor burden in BRCAMUT models. Clin Cancer Res; 23(12); 3097–108. ©2016 AACR.

Abstract A16: Potent and selective ATR inhibitors for the treatment of homologous-recombination deficient and PARPi-resistant cancers

Ataxia Telangiectasia and Rad3-related (ATR) and Checkpoint kinase 1 (CHK1) stabilize stalled replication forks and prevent their collapse into DNA double strand breaks (DSBs). Inhibition of ATR in cells experiencing oncogenic stress or harboring other cancer-associated defects synergistically increases the formation of DSBs and causes synthetic lethality. Thus specific targeting of ATR represents an emerging strategy to treat a broad spectrum of cancers, most notably those that currently lack effective treatments. We and others have shown that inhibition of the ATR checkpoint kinase is synthetically lethal with multiple distinct cancer-associated mutations, including p53 loss, oncogenic stress (HRAS-G12V, KRAS-G12D, NRAS-G12D, MYC and CCNE1), deficiency in homologous recombination (BRCA1/2, PALB2, ATM loss), alternative lengthening of telomeres (ALT), chromatin modification (SETD2 loss), and others. The specificity of ATR inhibitors is vital to their successful clinical application, since off-targeting increases toxicity to normal cells and limits value in personalized treatments. We report the generation of a novel class of highly potent and specific ATR inhibitors (ATRN series) that exhibit low nanomolar activity in cultured cells (IC50 = 2-8 nM) and do not detectably inhibit ATM, DNA-PK or mTOR (IC50 > 10 µM). In side-by-side comparisons, both the potency and selectivity of the ATRN series is superior to previously reported ATR inhibitors (VE821, VE822, AZD20, AZD6738, ETP-46464). In addition, the ATRN series has sufficient bioavailability and stability for in vivo application. Our lead compound (ATRN-119) slows progression of human BRCA2-deficient PDAC (CAPAN1) and RAS oncogene-driven p53-null colon tumors in mice with minimal toxicity to tissues under normal proliferative control, including the bone marrow and intestine. Additionally, mice engrafted with BRCA2-mutant patient-derived xenograft (PDX) ovarian tumors show a significant reduction in tumor progression after 5 weeks treatment of ATRN-119, and display no toxicity or significant weight loss. In addition, cancers that maintain or reacquire HR function, and thus are resistant to treatment with PARPi or cisplatin, remain responsive to ATRN series inhibitors. Thus, ATRN-119 is highly efficacious in suppressing tumor growth in multiple murine models, including suppression of patient-derived BRCA2-mutant ovarian, and PARP resistant cancers, suggesting that the clinical application of the ATRN series will provide a new and effective treatment for human malignancies with fewer side effects than conventional chemotherapies. In summary, the ATRN series is a highly selective and potent class of ATR inhibitors with therapeutic potential for treating a broad range of cancers. The potentially identified biomarkers, in particular HR-deficiency, will inform patient selection (a critical component for delivering medical benefit) for treatment with our agent and could reduce future clinical risk and side-effects. Thus targeting patients with HR-deficient cancers is a particularly promising strategy in treating a broad range of cancers. Citation Format: Laura R. Butler, Ryan L. Ragland, Hank J. Breslin, Erin George, Tina Gill, Matthew Scheiwer, Nicolas Gordon, Karen Knudsen, Fiona Simpkins, Oren Gilad and Eric J. Brown. Potent and selective ATR inhibitors for the treatment of homologous-recombination deficient and PARPi-resistant cancers [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 A16.

Abstract A02: A novel orthotopic ovarian patient derived xenograft model platform to investigate novel therapies for BRCA deficient ovarian cancers

Introduction: To create a personalized, targeted approach to high grade serous ovarian cancers (HGSOC), reliable preclinical models are essential. About ~50% of HGSOC have defects in genes involved in homologous recombination (HR) such as BRCA. PARP inhibitors (PARPi) capitalize on synthetic lethality in HR-deficient tumors, however, clinical efficacy is limited (response rate only ~40%). Patient derived xenografts (PDXs) are emerging as a reliable preclinical model that recapitulates principal characteristics of the patients9 tumor while remaining biologically stable while passaged in mice. We developed a BRCA1/2 orthotopic PDX experimental platform to study alternative strategies for synthetic lethality. We hypothesized that targeting the ATR/CHK1 axis is synthetically lethal in BRCA mutant HGSOC models. Experimental Procedures: Fresh HGSOC tumor was transplanted orthotopically to the fallopian tube/ovary of NSG 5-8 wk mice. Tumor growth was followed. Tumors were evaluated by IHC, genomic and proteomic analysis. Alu II probe staining was used to evaluate human stroma content. DNA sequencing was performed using a 153 OVCA gene panel. Reverse Phase Protein Array Analysis (RPPA) was evaluated for signaling pathway activation. Primary ovarian tumor cultures were developed from patients9 tumor for mechanistic studies. To study the ATR/CHK1 axis in HR-deficient HGSOC, PARPi (Olaparib), CHK1 inhibitor (CHK1i, MK8776), and ATR inhibitor (ATRi, AZD6738) were evaluated. PEO1 (BRCA2 mutant), PEO4 (BRCA wildtype) and JHOS4 (BRCA1 mutant) HGSOC cells were evaluated for cell proliferation, survival, and genome stability before and after treatment. BRCA2 mutant (8945delAA) PDX (WO-2-1) was expanded in 70 mice. Mice were randomized into 5 gps: untreated, carboplatin, PARPi, CHK1i, and ATRi. Treatment was initiated when tumors were 70-100mm3 and volume was assessed weekly with ultrasound. PARP tumor activity and response to PARPi was assessed with a PET PARP1 radiotracer [18F]FTT (fluorthanatrace). Results: We developed a pipeline to study HR deficient HGSOC. We created an orthotopic PDX platform from 15 BRCA mutant patients in order to accurately study OVCA tumorigenicity and metastasis in the native environment with a 90% take rate in generating tumors in mouse passage 1 (MP1), and 100% take rate for MP2 and MP3. The PDX model (WO-2-1) was similarly platinum sensitive as the patient after platinum treatment. Tumors were evaluated by genomic and proteomic analysis to identify a target population and streamline therapeutic approaches. Pathogenic mutation profiles from the original patient tumor were preserved in PDXs serially passaged (MP1-3). High pCHK1 (s345) was used as a marker for investigation of ATR/CHK1 inhibition in BRCA mutant PDX models. We showed that ATRi and CHK1i are similarly effective to PARPi in a BRCA2 mutant PDX. A novel PET PARP1 radiotracer [18F]FTT was used and demonstrated co-localization of signal in a BRCA2 mutant PDX, which was diminished with olaparib treatment. Conclusions: Although technically more challenging, the orthotopic transplantation technique is feasible in generating HGSOC PDX models with a high success rate that more closely resembles the natural environment for HGSOC progression. Evaluation of genomic and proteomic profiles of a tumor allows one to streamline targeted therapies for testing in PDX preclinical trials that may in the future be translated back to the patient. Citation Format: Erin George, Hyoung Kim, Janos Tanyi, Ryan Ragland, Eric Brown, Rugang Zhang, Patricia Brafford, Katrin Sproesser, Marilda Beqiri, Adina Vultur, Clemens Krepler, Brandon Weis, Kate Nathanson, Yuling Lu, Gordon Mills, Mehran Makvandi, Robert Mach, Mark Morgan, Fiona Simpkins. A novel orthotopic ovarian patient derived xenograft model platform to investigate novel therapies for BRCA deficient ovarian cancers. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr A02.

Abstract 4835: Targeting the ATR/CHK1 axis in combination with PARP inhibition is more effective than PARP inhibition alone in BRCA mutant models

Introduction: Approximately 50% of high grade serous ovarian cancers (HGSOC) have defects in genes involved in homologous recombination repair (HR). BRCA1/2 mutant HGSOCs have a deficiency in the repair of double strand DNA breaks by HR. Poly(ADP-ribose) polymerase inhibitors (PARPi) block the repair of single-stranded breaks leading to double strand DNA breaks which cannot be repaired efficiently in BRCA-deficient cancers capitalizing on synthetic lethality. PARPi have a modest clinical response of only ∼40% in recurrent BRCA mutant HGSOCs. We hypothesize that PARPi alone increases reliance on other DNA repair pathways such as ATR/CHK1 in HR deficient cells, and by targeting ATR/CHK1 in combination with PARPi would be more effective in eradicating tumor growth. Experimental Procedures: Effects of PARP inhibitor (PARPi, Olaparib), CHK1 inhibitor (CHK1i, MK8776), and ATR inhibitor (ATRi, AZD6738) on cell cycle, survival, colony formation, genome stability were evaluated in PEO1 (BRCA2 mutant), PEO4 (BRCA wild-type), JHOS4 (BRCA1 mutant), and WO-24 (BRCA wild-type) ovarian cancer cells. A BRCA2 mutant (8945delAA) orthotopic PDX model was used to evaluate PARPi alone or in combination with CHK1/ATRi. Targeted capture massively parallel sequencing, Reverse-Phase Protein Array Analysis (RPPA) and IHC were performed on cells and xenografts to evaluate for biomarkers of response. Results: Monotherapy with PARPi, CHK1i, and ATRi in vitro demonstrated selectivity in mediating cell death and DNA damage in BRCA1/2 mutant cell lines (PEO1, JHOS4) compared to BRCA1/2 wild-type, platinum resistant cell lines (PEO4, WO-24). However, monotherapy only results in ∼40-50% cell death in BRCA1/2 mutant cell lines. PARPi alone resulted in tumor suppression but not tumor eradication in a BRCA2 mutant PDX model. PARPi treatment resulted in an increase in ATR/CHK1 signaling in BRCA1/2 mutant cells. Treatment with ATR/CHK1i in combination with PARPi is synergistic in reducing survival of BRCA1/2 cells. Combination treatment was more effective in targeting cell cycle mediators, and promoting apoptosis. Treatment with either PARPi+ATRi or PARPi+CHK1i combinations was synergistic in causing tumor suppression but PARPi/ATRi combination caused tumor regression in a BRCA2 mutant PDX model. Conclusions: Strategies to optimize approaches capitalizing on synthetic lethality are needed for HR deficient HGSOC. PARPi is effective in BRCA deficient cancers but can potentially be more effective when combined with ATR/CHK1i. Citation Format: Erin George, Hyoung Kim, Janos Tanyi, Ryan Ragland, Rugang Zhang, Patricia Bradford, Clemens Krepler, Katherine Nathanson, Brandon Wenz, Yiling Lu, Gordon Mills, Mark Morgan, Fiona Simpkins. Targeting the ATR/CHK1 axis in combination with PARP inhibition is more effective than PARP inhibition alone in BRCA mutant models. [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 4835.

Abstract 1226: Highly specific ATR inhibitors as a therapeutic approach for a broad spectrum of cancers

Ataxia Telangiectasia and Rad3-related (ATR) and Checkpoint kinase 1 (CHK1) stabilize stalled replication forks and prevent their collapse into DNA double strand breaks (DSBs). Inhibition of ATR in cells experiencing oncogenic stress or harboring other cancer-associated defects synergistically increases the formation of DSBs and causes synthetic lethality. Thus specific targeting of ATR represents an emerging strategy to treat a broad spectrum of cancers, most notably those that currently lack effective treatments. Atrin Pharmaceuticals has synthesized a novel series of small molecules that inhibit ATR at low nanomolar concentrations in cultured cells. These compounds have the highest known potency for inhibiting ATR and maintain >800-fold lower cellular activity towards other kinases of the same family (ATM, DNA-PKcs and mTOR), which are substantially off-targeted by previously reported ATR inhibitors. Atrin9s lead compound (ATRN-119), as a single agent, selectively kills cancer cells subjected to oncogenic stress, alternative lengthening of telomeres (ALT) or loss of double strand break (DSB) homologous recombination repair mechanism (BRCA1 or BRCA2 deficiency). Furthermore, ATRN-119 cytotoxicity is synergistically enhanced when combined with conventional chemotherapeutics, such as etoposide, cisplatin and olaparib. Therefore, this inhibitor can be used in combination with other therapeutics to potentiate its anti-tumor activity. In vivo studies of RAS oncogene driven HCT116 p53-null flank tumors in mice demonstrate the ability of our lead compound to slow tumor progression, with minimal toxicity to tissues under normal proliferative control, including the bone marrow and intestine. Additionally, mice engrafted with BRCA2-mutant patient-derived ovarian tumors show a significant reduction in progression after 5 weeks treatment (100 mg/kg BID) of ATRN-119, and display no toxicity or significant weight loss. Thus, ATRN-119 is highly efficacious in suppressing tumor growth in multiple murine models, including suppression of patient-derived BRCA2-mutant ovarian cancer, suggesting that the clinical application of the ATRN series will provide a new and effective treatment for human malignancies with fewer side effects than conventional chemotherapies. Citation Format: Laura R. Butler, Ryan L. Ragland, Hank J. Breslin, Tina Gill, Erin George, Fiona Simpkins, Eric J. Brown, Oren Gilad. Highly specific ATR inhibitors as a therapeutic approach for a broad spectrum of cancers. [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 1226.