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Supplementary Figure 1

Supplementary Information

U87/Centrin2-EGFP/H2B-mCherry/puro cells were seeded on dishes with cover slip bottoms, treated with AZ32 (3 uM), and irradiated (5 Gy). Time-lapse videos were recorded intermittently (every 7 min) over 16 hrs. Treatment results in 11% aberrant mitoses (see Fig. 3F).

Primers used for targeted gene profiling on selected RAS/MAPK pathway genes either on Fluidigm or by qRT-PCR on Roche Lightcycler 480

Supplementary Figure 3

Supplementary Figure 2

KRAS mutation status of cell lines included in in vitro combination screen

The RAS-regulated RAF–MEK1/2–ERK1/2 (RAS/MAPK) signaling pathway is a major driver in oncogenesis and is frequently dysregulated in human cancers, primarily by mutations in BRAF or RAS genes. The clinical benefit of inhibitors of this pathway as single agents has only been realized in BRAF-mutant melanoma, with limited effect of single-agent pathway inhibitors in KRAS-mutant tumors. Combined inhibition of multiple nodes within this pathway, such as MEK1/2 and ERK1/2, may be necessary to effectively suppress pathway signaling in KRAS-mutant tumors and achieve meaningful clinical benefit. Here, we report the discovery and characterization of AZD0364, a novel, reversible, ATP-competitive ERK1/2 inhibitor with high potency and kinase selectivity. In vitro, AZD0364 treatment resulted in inhibition of proximal and distal biomarkers and reduced proliferation in sensitive BRAF-mutant and KRAS-mutant cell lines. In multiple in vivo xenograft models, AZD0364 showed dose- and time-dependent modulation of ERK1/2-dependent signaling biomarkers resulting in tumor regression in sensitive BRAF- and KRAS-mutant xenografts. We demonstrate that AZD0364 in combination with the MEK1/2 inhibitor, selumetinib (AZD6244 and ARRY142886), enhances efficacy in KRAS-mutant preclinical models that are moderately sensitive or resistant to MEK1/2 inhibition. This combination results in deeper and more durable suppression of the RAS/MAPK signaling pathway that is not achievable with single-agent treatment. The AZD0364 and selumetinib combination also results in significant tumor regressions in multiple KRAS-mutant xenograft models. The combination of ERK1/2 and MEK1/2 inhibition thereby represents a viable clinical approach to target KRAS-mutant tumors.

Supplementary Figure 6

U87/shp53/Centrin2-EGFP/H2B-mCherry/shp53 cells were seeded on dishes with cover slip bottoms and irradiated (5 Gy). Time-lapse videos were recorded intermittently (every 7 min) over 16 hrs. Treatment results in 12% aberrant mitoses (see Fig. 3F).

Supplementary materials and methods

U87/Centrin2-EGFP/H2B-mCherry/puro cells were seeded on dishes with cover slip bottoms and irradiated (5 Gy). Time-lapse videos were recorded intermittently (every 7 min) over 16 hrs. Treatment results in 2.7% aberrant mitoses (see Fig. 3F).

Supplementary Figure 5

Inhibition of ataxia-telangiectasia mutated (ATM) during radiotherapy of glioblastoma multiforme (GBM) may improve tumor control by short-circuiting the response to radiation-induced DNA damage. A major impediment for clinical implementation is that current inhibitors have limited central nervous system (CNS) bioavailability; thus, the goal was to identify ATM inhibitors (ATMi) with improved CNS penetration. Drug screens and refinement of lead compounds identified AZ31 and AZ32. The compounds were then tested in vivo for efficacy and impact on tumor and healthy brain. Both AZ31 and AZ32 blocked the DNA damage response and radiosensitized GBM cells in vitro. AZ32, with enhanced blood–brain barrier (BBB) penetration, was highly efficient in vivo as radiosensitizer in syngeneic and human, orthotopic mouse glioma model compared with AZ31. Furthermore, human glioma cell lines expressing mutant p53 or having checkpoint-defective mutations were particularly sensitive to ATMi radiosensitization. The mechanism for this p53 effect involves a propensity to undergo mitotic catastrophe relative to cells with wild-type p53. In vivo, apoptosis was >6-fold higher in tumor relative to healthy brain after exposure to AZ32 and low-dose radiation. AZ32 is the first ATMi with oral bioavailability shown to radiosensitize glioma and improve survival in orthotopic mouse models. These findings support the development of a clinical-grade, BBB-penetrating ATMi for the treatment of GBM. Importantly, because many GBMs have defective p53 signaling, the use of an ATMi concurrent with standard radiotherapy is expected to be cancer-specific, increase the therapeutic ratio, and maintain full therapeutic effect at lower radiation doses. Mol Cancer Ther; 17(8); 1637–47. ©2018 AACR.

Cell lines defined as sensitive to AZD0364 from 747 cell panel screen

Response of 747 cell lines to treatment with AZD0364 for 72 hours.

Supplementary Figure Legends

The RAS-regulated RAF–MEK1/2–ERK1/2 (RAS/MAPK) signaling pathway is a major driver in oncogenesis and is frequently dysregulated in human cancers, primarily by mutations in BRAF or RAS genes. The clinical benefit of inhibitors of this pathway as single agents has only been realized in BRAF-mutant melanoma, with limited effect of single-agent pathway inhibitors in KRAS-mutant tumors. Combined inhibition of multiple nodes within this pathway, such as MEK1/2 and ERK1/2, may be necessary to effectively suppress pathway signaling in KRAS-mutant tumors and achieve meaningful clinical benefit. Here, we report the discovery and characterization of AZD0364, a novel, reversible, ATP-competitive ERK1/2 inhibitor with high potency and kinase selectivity. In vitro, AZD0364 treatment resulted in inhibition of proximal and distal biomarkers and reduced proliferation in sensitive BRAF-mutant and KRAS-mutant cell lines. In multiple in vivo xenograft models, AZD0364 showed dose- and time-dependent modulation of ERK1/2-dependent signaling biomarkers resulting in tumor regression in sensitive BRAF- and KRAS-mutant xenografts. We demonstrate that AZD0364 in combination with the MEK1/2 inhibitor, selumetinib (AZD6244 and ARRY142886), enhances efficacy in KRAS-mutant preclinical models that are moderately sensitive or resistant to MEK1/2 inhibition. This combination results in deeper and more durable suppression of the RAS/MAPK signaling pathway that is not achievable with single-agent treatment. The AZD0364 and selumetinib combination also results in significant tumor regressions in multiple KRAS-mutant xenograft models. The combination of ERK1/2 and MEK1/2 inhibition thereby represents a viable clinical approach to target KRAS-mutant tumors.

Purpose: Combining radiotherapy (RT) with DNA damage response inhibitors may lead to increased tumor cell death through radiosensitization. DNA-dependent protein kinase (DNA-PK) plays an important role in DNA double-strand break repair via the nonhomologous end joining (NHEJ) pathway. We hypothesized that in addition to a radiosensitizing effect from the combination of RT with AZD7648, a potent and specific inhibitor of DNA-PK, combination therapy may also lead to modulation of an anticancer immune response. Experimental Design: AZD7648 and RT efficacy, as monotherapy and in combination, was investigated in fully immunocompetent mice in MC38, CT26, and B16-F10 models. Immunologic consequences were analyzed by gene expression and flow-cytometric analysis. Results: AZD7648, when delivered in combination with RT, induced complete tumor regressions in a significant proportion of mice. The antitumor efficacy was dependent on the presence of CD8+ T cells but independent of NK cells. Analysis of the tumor microenvironment revealed a reduction in T-cell PD-1 expression, increased NK-cell granzyme B expression, and elevated type I IFN signaling in mice treated with the combination when compared with RT treatment alone. Blocking of the type I IFN receptor in vivo also demonstrated a critical role for type I IFN in tumor growth control following combined therapy. Finally, this combination was able to generate tumor antigen-specific immunologic memory capable of suppressing tumor growth following rechallenge. Conclusions: Blocking the NHEJ DNA repair pathway with AZD7648 in combination with RT leads to durable immune-mediated tumor control.

Purpose: Literature regarding the WHO’s International Classification of Function, Disability and Health (ICF) has called for research into psychosocial adjustment processes. This project aims to establish the relevance of the Integrative Model of Adjustment to Chronic Conditions (IMACC) as a framework for research and a clinical tool in rehabilitation by linking it with the ICF. Methods: The study employed secondary analysis of data from the original IMACC grounded theory study, where 8 women and 2 men with type 2 diabetes mellitus participated. IMACC consists of 3 interconnected parts comprising a total of 13 components. Datasets used for the study consisted of the qualitative data underpinning each IMACC component. Meaningful concepts from each dataset were linked to ICF categories using the updated ICF linking rules. Results: Results showed that all 13 IMACC components accommodate ICF category codes from all health and health related ICF components in patterns consistent with the theoretical conceptualisation of each separate IMACC component. Conclusion: IMACC maps comprehensively to the ICF framework and provides a framework that may be useful for future ICF related research into biopsychosocial processes in psychosocial adjustment. IMACC provides a clinically applicable intervention for people with psychosocial adjustment difficulties consistent with the ICF framework.

The RAS-regulated RAF-MEK1/2-ERK1/2 (RAS/MAPK) signaling pathway is a major driver in oncogenesis and is frequently dysregulated in human cancers, primarily by mutations in

The RAS/MAPK pathway is a major driver of oncogenesis and is dysregulated in approximately 30% of human cancers, primarily by mutations in the BRAF or RAS genes. The extracellular-signal-regulated kinases (ERK1 and ERK2) serve as central nodes within this pathway. The feasibility of targeting the RAS/MAPK pathway has been demonstrated by the clinical responses observed through the use of BRAF and MEK inhibitors in BRAF V600E/K metastatic melanoma; however, resistance frequently develops. Importantly, ERK1/2 inhibition may have clinical utility in overcoming acquired resistance to RAF and MEK inhibitors, where RAS/MAPK pathway reactivation has occurred, such as relapsed BRAF V600E/K melanoma. We describe our structure-based design approach leading to the discovery of AZD0364, a potent and selective inhibitor of ERK1 and ERK2. AZD0364 exhibits high cellular potency (IC50 = 6 nM) as well as excellent physicochemical and absorption, distribution, metabolism, and excretion (ADME) properties and has demonstrated encouraging antitumor activity in preclinical models.

DNA-dependent protein kinase (DNA-PK) is a critical player in the DNA damage response (DDR) and instrumental in the non-homologous end-joining pathway (NHEJ) used to detect and repair DNA double-strand breaks (DSBs). We demonstrate that the potent and highly selective DNA-PK inhibitor, AZD7648, is an efficient sensitizer of radiation- and doxorubicin-induced DNA damage, with combinations in xenograft and patient-derived xenograft (PDX) models inducing sustained regressions. Using ATM-deficient cells, we demonstrate that AZD7648, in combination with the PARP inhibitor olaparib, increases genomic instability, resulting in cell growth inhibition and apoptosis. AZD7648 enhanced olaparib efficacy across a range of doses and schedules in xenograft and PDX models, enabling sustained tumour regression and providing a clear rationale for its clinical investigation. Through its differentiated mechanism of action as an NHEJ inhibitor, AZD7648 complements the current armamentarium of DDR-targeted agents and has potential in combination with these agents to achieve deeper responses to current therapies., DNA-dependent protein kinase (DNA-PK) plays a major role in the DNA damage response upon double-strand break formation. Here, the authors show that the DNA-PK inhibitor AZD7648, enhances the activity of radiotherapy, chemotherapy and the PARP inhibitor olaparib in multiple mouse tumour models.

Inhibition of ataxia-telangiectasia mutated (ATM) during radiotherapy of glioblastoma multiforme (GBM) may improve tumor control by short-circuiting the response to radiation-induced DNA damage. A major impediment for clinical implementation is that current inhibitors have limited central nervous system (CNS) bioavailability; thus, the goal was to identify ATM inhibitors (ATMi) with improved CNS penetration. Drug screens and refinement of lead compounds identified AZ31 and AZ32. The compounds were then tested in vivo for efficacy and impact on tumor and healthy brain. Both AZ31 and AZ32 blocked the DNA damage response and radiosensitized GBM cells in vitro. AZ32, with enhanced blood–brain barrier (BBB) penetration, was highly efficient in vivo as radiosensitizer in syngeneic and human, orthotopic mouse glioma model compared with AZ31. Furthermore, human glioma cell lines expressing mutant p53 or having checkpoint-defective mutations were particularly sensitive to ATMi radiosensitization. The mechanism for this p53 effect involves a propensity to undergo mitotic catastrophe relative to cells with wild-type p53. In vivo, apoptosis was >6-fold higher in tumor relative to healthy brain after exposure to AZ32 and low-dose radiation. AZ32 is the first ATMi with oral bioavailability shown to radiosensitize glioma and improve survival in orthotopic mouse models. These findings support the development of a clinical-grade, BBB-penetrating ATMi for the treatment of GBM. Importantly, because many GBMs have defective p53 signaling, the use of an ATMi concurrent with standard radiotherapy is expected to be cancer-specific, increase the therapeutic ratio, and maintain full therapeutic effect at lower radiation doses. Mol Cancer Ther; 17(8); 1637–47. ©2018 AACR.

ERK1/2 is a key protein in the MAPK pathway, regulating phenotypes such as proliferation and migration. Upstream mutations (e.g., KRAS mutations in non-small-cell lung (NSCLC)) can cause the pathway to become constitutively activated, driving tumor growth. AZD0364 is a potent, selective inhibitor of ERK's kinase activity against its cytosolic substrate p90RSK. It is currently in preclinical development, where it has shown dose-dependent, anti-tumor activity in xenograft models of KRAS-mutant NSCLC, including Calu-6 (where it shows regression) and A549. Treatment with AZD0364 demonstrates rapid and near-complete inhibition of phospho-p90RSK. In addition, prolonged inhibition with AZD0364 causes a gradual downregulation of p90RSK protein over time, without any corresponding change in p90RSK mRNA. Here we present a pharmacokinetic/pharmacodynamic (PK/PD) model that links AZD0364 concentration to inhibition of ERK activity through both a direct inhibition of phospho-p90RSK and an indirect down-regulation of total-p90RSK protein. Anti-proliferative and pro-apoptotic effects on efficacy are linked to changes in p90RSK. The model leads to two key implications (i) repeated dosing will cause apparent potency to improve over time, since the pool of available substrate (i.e., p90RSK) is itself being reduced and (ii) recovery of signaling to baseline will depend not on washout of the inhibitor but on protein synthesis rates. Protein half-lives appear quite different between tumor models of KRAS-mutant NSCLC, with A549 (~20h) significantly slower than Calu-6 (~4h). The model provides a conceptual framework on which to link the timescale of PD changes with those seen in efficacy. Taken together, this means that while a new PD steady-state is achieved in Calu-6 in a few days, it also recovers quickly, necessitating constant cover (daily dosing) to drive regression. On the other hand, while A549 is more robust to inhibition, and slower to reach steady-state inhibition (~2 weeks), it is also slower to recover, so that intermittent schedules can achieve efficacy similar to those achievable with daily dosing. Citation Format: Francis D. Gibbons, Linda Sandin, Lyndsey Hanson, Rebecca Whiteley, Paul Farrington, Nicola Lindsay, Emma Davies, J Elizabeth Pease, Vikki Flemington. A PK/PD model quantitatively describes inhibition and down-regulation of p90RSK by ERK inhibitor AZD0364 [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 4913.