Epitope Lability of Phosphorylated Biomarkers of the DNA Damage Response Pathway Results in Increased Vulnerability to Effects of Delayed or Incomplete Formalin Fixation.

Phosphorylated biomarkers are crucial for our understanding of drug mechanism of action and dose selection during clinical trials, particularly for drugs that target protein kinases, such as DNA-damage-response (DDR) inhibitors. However, tissue fixation conditions needed to preserve DDR-specific phospho-biomarkers have not been previously investigated. Using xenograft tissues and tightly controlled formalin fixation conditions, we assessed how preanalytical factors affect phosphorylated DDR biomarkers pRAD50(Ser635), ɣH2AX(Ser139), pKAP1(Ser824), and non-phosphorylated biomarkers cMYC and ATM. Cold ischemia times ranged from 15 min to 6 hr, and the fixation duration ranged from 24 hr to 4 weeks. Epitopes pRAD50 and pKAP1 appeared the most labile assessed with staining loss after just 15 min of cold ischemia time, while ATM was more robust showing consistent expression up to 1 hr of cold ischemia. Notably, ɣH2AX expression was lost with formalin fixation over 48 hr. The use of core needle biopsies where possible and novel fixation methods such as the 2-step temperature-controlled formalin approach may improve phosphorylated biomarker preservation; however, practical challenges may affect wider clinical application. The most essential tissue-processing step when downstream analysis includes DDR phosphorylated biomarkers is immediate tissue submersion in formalin, without delay, upon excision from the patient, followed by room temperature fixation for 24 hr.

Relevance of ATM Status in Driving Sensitivity to DNA Damage Response Inhibitors in Patient-Derived Xenograft Models.

Ataxia-telangiectasia mutated gene (ATM) is a key component of the DNA damage response (DDR) and double-strand break repair pathway. The functional loss of ATM (ATM deficiency) is hypothesised to enhance sensitivity to DDR inhibitors (DDRi). Whole-exome sequencing (WES), immunohistochemistry (IHC), and Western blotting (WB) were used to characterise the baseline ATM status across a panel of ATM mutated patient-derived xenograft (PDX) models from a range of tumour types. Antitumour efficacy was assessed with poly(ADP-ribose)polymerase (PARP, olaparib), ataxia- telangiectasia and rad3-related protein (ATR, AZD6738), and DNA-dependent protein kinase (DNA-PK, AZD7648) inhibitors as a monotherapy or in combination to associate responses with ATM status. Biallelic truncation/frameshift ATM mutations were linked to ATM protein loss while monoallelic or missense mutations, including the clinically relevant recurrent R3008H mutation, did not confer ATM protein loss by IHC. DDRi agents showed a mixed response across the PDX's but with a general trend toward greater activity, particularly in combination in models with biallelic ATM mutation and protein loss. A PDX with an ATM splice-site mutation, 2127T > C, with a high relative baseline ATM expression and KAP1 phosphorylation responded to all DDRi treatments. These data highlight the heterogeneity and complexity in describing targetable ATM-deficiencies and the fact that current patient selection biomarker methods remain imperfect; although, complete ATM loss was best able to enrich for DDRi sensitivity.

The DNA-PK Inhibitor AZD7648 Sensitizes Patient-Derived Ovarian Cancer Xenografts to Pegylated Liposomal Doxorubicin and Olaparib Preventing Abdominal Metastases.

Ovarian cancer is the deadliest gynecologic cancer, with a 5-year survival rate of 30%, when the disease has spread throughout the peritoneal cavity. We investigated the efficacy to delay disease progression by the DNA-dependent protein kinase (DNA-PK) inhibitor AZD7648, administered in combination with two of the therapeutic options for patient management: either pegylated liposomal doxorubicin (PLD) or the PARP inhibitor olaparib. Patient-derived ovarian cancer xenografts (OC-PDX) were transplanted subcutaneously to evaluate the effect of treatment on tumor growth, or orthotopically in the peritoneal cavity to evaluate the effect on metastatic spread. AZD7648 was administered orally in combination with PLD (dosed intravenously) or with olaparib (orally). To prove the inhibition of DNA-PK in the tumors, we measured pDNA-PKcs, pRPA32, and γH2AX, biomarkers of DNA-PK activity. AZD7648 enhanced the therapeutic efficacy of PLD in all the OC-PDXs tested, regardless of their BRCA status or sensitivity to cisplatin or PLD. The treatment caused disease stabilization, which persisted despite therapy discontinuation for tumors growing subcutaneously, and significantly impaired the abdominal metastatic dissemination, prolonging the lifespan of mice implanted orthotopically. AZD7648 potentiated the efficacy of olaparib in BRCA-deficient OC-PDXs but did not sensitize BRCA-proficient OC-PDXs to olaparib, despite an equivalent inhibition of DNA-PK, suggesting the need of a preexisting olaparib activity to benefit from the addition of AZD7648. This work suggests that AZD7648, an inhibitor of DNA-PK, dosed in combination with PLD or olaparib is an exciting therapeutic option that could benefit patients with ovarian cancer and should be explored in clinical trials.