The poly (ADP-ribose) polymerase (PARP) inhibitor olaparib is FDA approved for the treatment of BRCA-mutated breast, ovarian and pancreatic cancers. Olaparib inhibits PARP1/2 enzymatic activity and ...traps PARP1 on DNA at single-strand breaks, leading to replication-induced DNA damage that requires BRCA1/2-dependent homologous recombination repair. Moreover, DNA damage response pathways mediated by the ataxia-telangiectasia mutated (ATM) and ataxia-telangiectasia mutated and Rad3-related (ATR) kinases are hypothesised to be important survival pathways in response to PARP-inhibitor treatment. Here, we show that olaparib combines synergistically with the ATR-inhibitor AZD6738 (ceralasertib), in vitro, leading to selective cell death in ATM-deficient cells. We observe that 24 h olaparib treatment causes cells to accumulate in G2-M of the cell cycle, however, co-administration with AZD6738 releases the olaparib-treated cells from G2 arrest. Selectively in ATM-knockout cells, we show that combined olaparib/AZD6738 treatment induces more chromosomal aberrations and achieves this at lower concentrations and earlier treatment time-points than either monotherapy. Furthermore, single-agent olaparib efficacy in vitro requires PARP inhibition throughout multiple rounds of replication. Here, we demonstrate in several ATM-deficient cell lines that the olaparib and AZD6738 combination induces cell death within 1-2 cell divisions, suggesting that combined treatment could circumvent the need for prolonged drug exposure. Finally, we demonstrate in vivo combination activity of olaparib and AZD6738 in xenograft and PDX mouse models with complete ATM loss. Collectively, these data provide a mechanistic understanding of combined PARP and ATR inhibition in ATM-deficient models, and support the clinical development of AZD6738 in combination with olaparib.
DNA damage checkpoint kinases ATR and WEE1 are among key regulators of DNA damage response pathways protecting cells from replication stress, a hallmark of cancer that has potential to be exploited ...for therapeutic use. ATR and WEE1 inhibitors are in early clinical trials and success will require greater understanding of both their mechanism of action and biomarkers for patient selection. Here, we report selective antitumor activity of ATR and WEE1 inhibitors in a subset of non-germinal center B-cell (GCB) diffuse large B-cell lymphoma (DLBCL) cell lines, characterized by high MYC protein expression and
deletion. Activity correlated with the induction of replication stress, indicated by increased origin firing and retardation of replication fork progression. However, ATR and WEE1 inhibitors caused different amounts of DNA damage and cell death in distinct phases of the cell cycle, underlying the increased potency observed with WEE1 inhibition. ATR inhibition caused DNA damage to manifest as 53BP1 nuclear bodies in daughter G
cells leading to G
arrest, whereas WEE1 inhibition caused DNA damage and arrest in S phase, leading to earlier onset apoptosis.
xenograft DLBCL models confirmed differences in single-agent antitumor activity, but also showed potential for effective ATR inhibitor combinations. Importantly, insights into the different inhibitor mechanisms may guide differentiated clinical development strategies aimed at exploiting specific vulnerabilities of tumor cells while maximizing therapeutic index. Our data therefore highlight clinical development opportunities for both ATR and WEE1 inhibitors in non-GCB DLBCL subtypes that represent an area of unmet clinical need. SIGNIFICANCE: ATR and WEE1 inhibitors demonstrate effective antitumor activity in preclinical models of DLBCL associated with replication stress, but new mechanistic insights and biomarkers of response support a differentiated clinical development strategy.
In oncological drug development, animal studies continue to play a central role in which the volume of subcutaneous tumours is monitored to assess the efficacy of new drugs. The tumour volume is ...estimated by taking the volume to be that of a regular spheroid with the same dimensions. However, this method is subjective, insufficiently traceable, and is subject to error in the accuracy of volume estimates as tumours are frequently irregular.
This paper reviews the standard technique for tumour volume assessment, calliper measurements, by conducting a statistical review of a large dataset consisting of 2,500 tumour volume measurements from 1,600 mice by multiple operators across 6 mouse strains and 20 tumour models. Additionally, we explore the impact of six different tumour morphologies on volume estimation and the detection of treatment effects using a computational tumour growth model. Finally, we propose an alternative method to callipers for estimating volume-BioVolumeTM, a 3D scanning technique. BioVolume simultaneously captures both stereo RGB (Red, Green and Blue) images from different light sources and infrared thermal images of the tumour in under a second. It then detects the tumour region automatically and estimates the tumour volume in under a minute. Furthermore, images can be processed in parallel within the cloud and so the time required to process multiple images is similar to that required for a single image. We present data of a pre-production unit test consisting of 297 scans from over 120 mice collected by four different operators.
This work demonstrates that it is possible to record tumour measurements in a rapid minimally invasive, morphology-independent way, and with less human-bias compared to callipers, whilst also improving data traceability. Furthermore, the images collected by BioVolume may be useful, for example, as a source of biomarkers for animal welfare and secondary drug toxicity / efficacy.
PARP inhibitors are synthetically lethal with BRCA1/2 mutations, and in this setting, accumulation of DNA damage leads to cell death. Because increased DNA damage and subsequent immune activation can ...prime an anti-tumor immune response, we studied the impact of olaparib ± immune checkpoint blockade (ICB) on anti-tumor activity and the immune microenvironment. Concurrent combination of olaparib, at clinically relevant exposures, with ICB gave durable and deeper anti-tumor activity in the Brca1m BR5 model vs. monotherapies. Olaparib and combination treatment modulated the immune microenvironment, including increases in CD8+ T cells and NK cells, and upregulation of immune pathways, including type I IFN and STING signaling. Olaparib also induced a dose-dependent upregulation of immune pathways, including JAK/STAT, STING and type I IFN, in the tumor cell compartment of a BRCA1m (HBCx-10) but not a BRCA WT (HBCx-9) breast PDX model. In vitro, olaparib induced BRCAm tumor cell-specific dendritic cell transactivation. Relevance to human disease was assessed using patient samples from the MEDIOLA (NCT02734004) trial, which showed increased type I IFN, STING, and JAK/STAT pathway expression following olaparib treatment, in line with preclinical findings. These data together provide evidence for a mechanism and schedule underpinning potential benefit of ICB combination with olaparib.
The kinase ataxia telangiectasia mutated and rad3 related (ATR) is a key regulator of the DNA-damage response and the apical kinase which orchestrates the cellular processes that repair stalled ...replication forks (replication stress) and associated DNA double-strand breaks. Inhibition of repair pathways mediated by ATR in a context where alternative pathways are less active is expected to aid clinical response by increasing replication stress. Here we describe the development of the clinical candidate 2 (AZD6738), a potent and selective sulfoximine morpholinopyrimidine ATR inhibitor with excellent preclinical physicochemical and pharmacokinetic (PK) characteristics. Compound 2 was developed improving aqueous solubility and eliminating CYP3A4 time-dependent inhibition starting from the earlier described inhibitor 1 (AZ20). The clinical candidate 2 has favorable human PK suitable for once or twice daily dosing and achieves biologically effective exposure at moderate doses. Compound 2 is currently being tested in multiple phase I/II trials as an anticancer agent.
AZD6738 (ceralasertib) is a potent and selective orally bioavailable inhibitor of ataxia telangiectasia and Rad3-related (ATR) kinase. ATR is activated in response to stalled DNA replication forks to ...promote G2-M cell-cycle checkpoints and fork restart. Here, we found AZD6738 modulated CHK1 phosphorylation and induced ATM-dependent signaling (pRAD50) and the DNA damage marker γH2AX. AZD6738 inhibited break-induced replication and homologous recombination repair. In vitro sensitivity to AZD6738 was elevated in, but not exclusive to, cells with defects in the ATM pathway or that harbor putative drivers of replication stress such as CCNE1 amplification. This translated to in vivo antitumor activity, with tumor control requiring continuous dosing and free plasma exposures, which correlated with induction of pCHK1, pRAD50, and γH2AX. AZD6738 showed combinatorial efficacy with agents associated with replication fork stalling and collapse such as carboplatin and irinotecan and the PARP inhibitor olaparib. These combinations required optimization of dose and schedules in vivo and showed superior antitumor activity at lower doses compared with that required for monotherapy. Tumor regressions required at least 2 days of daily dosing of AZD6738 concurrent with carboplatin, while twice daily dosing was required following irinotecan. In a BRCA2-mutant patient-derived triple-negative breast cancer (TNBC) xenograft model, complete tumor regression was achieved with 3 to5 days of daily AZD6738 per week concurrent with olaparib. Increasing olaparib dosage or AZD6738 dosing to twice daily allowed complete tumor regression even in a BRCA wild-type TNBC xenograft model. These preclinical data provide rationale for clinical evaluation of AZD6738 as a monotherapy or combinatorial agent.
This detailed preclinical investigation, including pharmacokinetics/pharmacodynamics and dose-schedule optimizations, of AZD6738/ceralasertib alone and in combination with chemotherapy or PARP inhibitors can inform ongoing clinical efforts to treat cancer with ATR inhibitors.
CXCR2 has been suggested to have both tumor-promoting and tumor-suppressive properties. Here we show that CXCR2 signaling is upregulated in human pancreatic cancer, predominantly in ...neutrophil/myeloid-derived suppressor cells, but rarely in tumor cells. Genetic ablation or inhibition of CXCR2 abrogated metastasis, but only inhibition slowed tumorigenesis. Depletion of neutrophils/myeloid-derived suppressor cells also suppressed metastasis suggesting a key role for CXCR2 in establishing and maintaining the metastatic niche. Importantly, loss or inhibition of CXCR2 improved T cell entry, and combined inhibition of CXCR2 and PD1 in mice with established disease significantly extended survival. We show that CXCR2 signaling in the myeloid compartment can promote pancreatic tumorigenesis and is required for pancreatic cancer metastasis, making it an excellent therapeutic target.
Display omitted
•CXCR2 signaling is upregulated in myeloid cells in human pancreatic cancer•Cxcr2 loss reduces metastasis and inhibition prolongs tumor-free survival in mice•Neutrophils/MDSCs play a key role in the establishment of the metastatic niche•CXCR2 inhibition enhances T cell entry and confers sensitivity to anti-PD1 therapy
Steele et al. show that CXCR2 is important in immune modulation of pancreatic cancer and that inhibition of CXCR2 reduces metastasis and improves response to gemcitabine and anti-PD1. Peptide inhibitor, but not germline deletion of Cxcr2, improved survival, revealing differential effects in early and late tumors.
Magnetic resonance imaging (MRI) can measure the effects of therapies targeting the tumor vasculature and has demonstrated that vascular-damaging agents (VDA) induce acute vascular shutdown in tumors ...in human and animal models. However, at subtherapeutic doses, blood flow may recover before the induction of significant levels of necrosis. We present the relationship between changes in MRI biomarkers and tumor necrosis. Multiple MRI measurements were taken at 4.7 T in athymic rats (n = 24) bearing 1.94 ± 0.2-cm3 subcutaneous Hras5 tumors (ATCC 41000) before and 24 hours after clinically relevant doses of the VDA, ZD6126 (0-10 mg/kg, i.v.). We measured effective transverse relaxation rate (R2*), initial area under the gadolinium concentration-time curve (IAUGC60/150), equivalent enhancing fractions (EHF60/150), time constant (Ktrans), proportion of hypoperfused voxels as estimated from fit failures in Ktrans analysis, and signal intensity (SI) in T2-weighted MRI (T2W). ZD6126 treatment induced < 90% dose-dependent tumor necrosis at 10 mg/kg; correspondingly, SI changes were evident from T2W MRI. Although R2* did not correlate, other MRI biomarkers significantly correlated with necrosis at doses of ≥ 5 mg/kg ZD6126. These data on Hras5 tumors suggest that the quantification of hypoperfused voxels might provide a useful biomarker of tumor necrosis.
PDF
