Radiation in Combination with Immune Checkpoint Blockade and DNA Damage Response Inhibitors in Mice: Dosage Optimization in MC38 Syngeneic Tumors via Modelling and Simulation.

Clinical trials assessing the impact of radiotherapy (RT) in combination with DNA damage response pathway inhibitors (DDRis) and/or immune checkpoint blockade are currently ongoing. However, current methods for optimizing dosage and schedule are limited. A mathematical model was developed to capture the impacts of RT in combination with DDRi and/or anti-PD-L1 [immune checkpoint inhibitor (ICI)] on tumor immune interactions in the MC38 syngeneic tumor model. The model was fitted to datasets that assessed the impact of RT in combination with the DNA protein kinase inhibitor (DNAPKi) AZD7648. The model was further fitted to datasets from studies that were used to assess both RT/ICI combinations as well as RT/ICI combinations followed by concurrent administration of the poly ADP ribose polymerase inhibitor (PARPi) olaparib. Nonlinear mixed-effects modeling was performed followed by internal validation with visual predictive checks (VPC). Simulations of alternative dosage regimens and scheduling were performed to identify optimal candidate dosage regimens of RT/DNAPKi and RT/PARPi/ICI. Model fits and VPCs confirmed a successful internal validation for both datasets and demonstrated very small differences in the median, lower, and upper percentile values of tumor diameters between RT/ICI and RT/PARPi/ICI, which indicated that the triple combination of RT/PARPi/ICI at the given dosage and schedule does not provide additional benefit compared with ICI in combination with RT. Simulation of alternative dosage regimens indicated that lowering the dosage of ICI to between 2 and 4 mg/kg could induce similar benefits to the full dosage regimen, which could be of translational benefit. SIGNIFICANCE STATEMENT: This work provides a mixed-effects model framework to quantify the effects of combination radiotherapy/DNA damage response pathway inhibitors/immune checkpoint inhibitors in preclinical tumor models and identify optimal dosage regimens, which could be of translational benefit.

Mixed effects modeling of radiotherapy in combination with immune checkpoint blockade or inhibitors of the DNA damage response pathway.

Dosage optimization to maximize efficacy and minimize toxicity is a potential issue when administering radiotherapy (RT) in combination with immune checkpoint blockade (ICB) or inhibitors of the DNA Damage Response Pathway (DDRi) in the clinic. Preclinical models and mathematical modeling can help identify ideal dosage schedules to observe beneficial effects of a tri-therapy. The aim of this study is to describe a mathematical model to capture the impact of RT in combination with inhibitors of the DNA Damage Response Pathway or blockade of the immune checkpoint protein - programmed death ligand 1 (PD-L1). This model describes how RT mediated activation of antigen presenting cells can induce an increase in cytolytic T cells capable of targeting tumor cells, and how combination drugs can potentiate the immune response by inhibiting the rate of T cell exhaustion. The model was fitted using preclinical data, where MC38 tumors were treated in vivo with RT alone or in combination with anti-PD-L1 as well as with either olaparib or the ataxia telangiectasia mutated (ATM) inhibitor-AZD0156. The model successfully described the observed data and goodness-of-fit, using visual predictive checks also confirmed a successful internal model validation for each treatment modality. The results demonstrated that the anti-PD-L1 effect in combination with RT was maximal in vivo and any additional benefit of DDRi at the given dosage and schedule used was undetectable. Model fit results indicated AZD0156 to be a more potent DDRi than olaparib. Simulations of alternative doses indicated that reducing efficacy of anti-PD-L1 by 68% would potentially provide evidence for a benefit of ATM inhibition in combination with ICB and increase the relative efficacy of tri-therapy.

Inhibition of DNA-PK with AZD7648 Sensitizes Tumor Cells to Radiotherapy and Induces Type I IFN-Dependent Durable Tumor Control.

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.

AZD0364 Is a Potent and Selective ERK1/2 Inhibitor That Enhances Antitumor Activity in KRAS-Mutant Tumor Models when Combined with the MEK Inhibitor, Selumetinib.

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.

AZD7648 is a potent and selective DNA-PK inhibitor that enhances radiation, chemotherapy and olaparib activity.

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.

Discovery of a Potent and Selective Oral Inhibitor of ERK1/2 (AZD0364) That Is Efficacious in Both Monotherapy and Combination Therapy in Models of Nonsmall Cell Lung Cancer (NSCLC).

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.

Orally Bioavailable and Blood-Brain Barrier-Penetrating ATM Inhibitor (AZ32) Radiosensitizes Intracranial Gliomas in Mice.

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.