Precision Oncology with Drugs Targeting the Replication Stress, ATR, and Schlafen 11

Precision medicine aims to implement strategies based on the molecular features of tumors and optimized drug delivery to improve cancer diagnosis and treatment. DNA replication is a logical approach because it can be targeted by a broad range of anticancer drugs that are both clinically approved and in development. These drugs increase deleterious replication stress (RepStress); however, how to selectively target and identify the tumors with specific molecular characteristics are unmet clinical needs. Here, we provide background information on the molecular processes of DNA replication and its checkpoints, and discuss how to target replication, checkpoint, and repair pathways with ATR inhibitors and exploit Schlafen 11 (SLFN11) as a predictive biomarker.

Schlafen 11 (SLFN11), a putative predictive biomarker of platinum/PARPi response, is frequently detected on circulating tumor cells (CTCs) in advanced prostate cancer.

e17039 Background: Schlafen 11 (SLFN11) is a DNA repair protein (DNA/RNA helicase homology) that is recruited to stressed replication forks and leads to cell death. Recent ph II trial data in Extensive Stage Small-Cell Lung Cancer (ES-SCLC) [Byers et al. JCO 2018 PMID: 29906251] and a retrospective analysis of Circulating Tumor Cells (CTCs) and tumor tissue in patients (Pts) with advanced prostate cancer [Conteduca et al. Mol Cancer Therapeutics 2020 PMID: 32127465] suggested that SLFN11 expression predicts sensitivity to DNA damage targeting agents. In both contexts, metastatic tumor biopsies may not provide adequate material for profiling. We assessed the frequency of SLFN11 expression in CTCs isolated from blood in men with progressing metastatic Castration Resistant Prostate Cancer (mCRPC) and related expression to Homologous Recombination Repair (HRR) alterations identified in metastatic tumor biopsies profiled by MSK-IMPACT. Methods: 95 patients with progressing mCRPC about to start a new systemic therapy who had undergone pre-treatment metastatic tumor profiling by MSK-IMPACT and a matched blood draw for CTC profiling were selected. Blood was sent overnight to Epic Sciences and processed onto glass pathology slides and bio-banked until analysis. Detected CTCs (cytokeratin (CK) positive and leukocyte (CD45) negative) were analyzed for SLFN11 protein expression by immunofluorescence and correlated to Homologous Recombination Repair (HRR) alterations in metastatic biopsy from the bone, lymph node, or visceral metastases (15 PROFOUND genes). A mean of 1.2 mL of blood was analyzed per patient. Results: CTCs with SLFN11 expression (DAPI+, CK+, CD45-, SLFN11+) were detected in 28.4% (27/95) of the patient sample analyzed. SLFN11 signal was found to overlap with the nuclear DAPI signal. Individual CTC expression of SLFN11 in a sample was heterogeneous and ranged from a minimum of 2.9% to 100%. Seven of 10 (70%) of patients with a BRCA1/2 or ATM alteration had a least one SLFN11 expressing CTC. In contrast, in patients with other HRR alterations, only 20% (1/5) had CTCs with SLFN11. Sequencing of single CTCs is ongoing. Conclusions: SLFN11 expression is detected with high frequency in CTCs in men with progressing mCRPC. In whom CTCs are detected, the majority of patients with BRCA1/2 or ATM altered tumors also had SLFN11 expressing CTCs. The results support the prospective evaluation of CTC SLFN11 expression as a predictive biomarker for PARPi or platinum agents.

SLFN11 promotes CDT1 degradation by CUL4 in response to replicative DNA damage, while its absence leads to synthetic lethality with ATR/CHK1 inhibitors

Schlafen-11 (SLFN11) inactivation in ∼50% of cancer cells confers broad chemoresistance. To identify therapeutic targets and underlying molecular mechanisms for overcoming chemoresistance, we performed an unbiased genome-wide RNAi screen in SLFN11-WT and -knockout (KO) cells. We found that inactivation of Ataxia Telangiectasia- and Rad3-related (ATR), CHK1, BRCA2, and RPA1 overcome chemoresistance to camptothecin (CPT) in SLFN11-KO cells. Accordingly, we validate that clinical inhibitors of ATR (M4344 and M6620) and CHK1 (SRA737) resensitize SLFN11-KO cells to topotecan, indotecan, etoposide, cisplatin, and talazoparib. We uncover that ATR inhibition significantly increases mitotic defects along with increased CDT1 phosphorylation, which destabilizes kinetochore-microtubule attachments in SLFN11-KO cells. We also reveal a chemoresistance mechanism by which CDT1 degradation is retarded, eventually inducing replication reactivation under DNA damage in SLFN11-KO cells. In contrast, in SLFN11-expressing cells, SLFN11 promotes the degradation of CDT1 in response to CPT by binding to DDB1 of CUL4CDT2 E3 ubiquitin ligase associated with replication forks. We show that the C terminus and ATPase domain of SLFN11 are required for DDB1 binding and CDT1 degradation. Furthermore, we identify a therapy-relevant ATPase mutant (E669K) of the SLFN11 gene in human TCGA and show that the mutant contributes to chemoresistance and retarded CDT1 degradation. Taken together, our study reveals new chemotherapeutic insights on how targeting the ATR pathway overcomes chemoresistance of SLFN11-deficient cancers. It also demonstrates that SLFN11 irreversibly arrests replication by degrading CDT1 through the DDB1–CUL4CDT2 ubiquitin ligase.