Bose S. Kochupurakkal

Purpose: To determine the maximum tolerated dose (MTD) of the pan–class I PI3K inhibitor buparlisib (B) in combination with weekly cisplatin and radiotherapy (RT) among patients with locally advanced (LA) squamous cell cancer of the head and neck (SCCHN) and tobacco history. Patients and Methods: Patients with stage III/IV LA-SCCHN [American Joint Committee on Cancer (AJCC), Seventh Edition] and >10 pack-year tobacco use history were enrolled. Patients received B during a 2-week run-in phase and during chemoradiotherapy (CRT) consisting of standard 70 Gy of RT plus weekly cisplatin. An exploratory analysis of genomic sequencing was performed on biopsy specimens. Results: Twenty-three patients were enrolled {n = 17 at the MTD [B 40 mg daily and cisplatin (CDDP) 30 mg/m2/week]}. Ninety-one percent (21/23) had stage IV disease. Human papillomavirus (HPV) was detected in 15 of 18 cases with oral/oropharyngeal disease. The median follow-up was 23 months. Recurrence was detected in five patients among whom four had evaluable tumor samples. Fifty-percent (2/4) of patients with evaluable tumor samples were characterized by activation of the PI3K pathway. In five patients whose disease responded during the 2-week run-in phase with B alone, 3 of 4 with sequencing data showed loss-of-function mutations in either tumor necrosis factor receptor–associated factor 3 (TRAF3) or cylindromatosis lysine deubiquitinase (CYLD). Preclinical studies with mutations in TRAF3 or CYLD modeled via CRISPR/Cas9 knockout in HPV+ SCCHN cells demonstrate that loss of TRAF3 or CYLD may sensitize SCCHN cell lines to PI3K through mechanisms other than blocking the NF-κB pathway. Conclusions: Low-dose B with CRT was feasible and active in this high-risk cohort, although escalation to the standard weekly cisplatin dose of 40 mg/m2 was not possible. Our data suggest that TRAF3/CYLD-mutant SCCHN may be susceptible to PI3K inhibition with B, whereas PI3K pathway activation seemed to be associated with poor outcomes in this limited dataset.

ATR inhibition has been proposed as a strategy for tumors under replication stress or those harboring ATM deficiency. However, anti-tumor activity is limited by DNA-PK and ATM compensation. Here, we have delineated distinct roles for DNA-PK and ATM during ATR inhibitor exposure. Both kinases are activated in a cell cycle-dependent manner at the S/G2 boundary following ATR inhibition. DNA-PK imposes the S/G2 checkpoint and promotes completion of DNA replication, whereas ATM detects persistent aberrant DNA replication structures with associated DNA damage in late S and G2/M to establish a G2/M checkpoint promoting DNA repair. In ATM-deficient cells and patient tumors, DNA-PK is activated following ATR inhibition and assumes greater versatility by not only enforcing the S/G2 checkpoint but also by detecting aberrant replication structures in S phase in place of ATM, allowing cells to tolerate ATR inhibition. Consequently, DNA-PK inhibition synergizes with ATR inhibition in ATM-deficient cells and enhances ATR inhibitor efficacy in ATM-deficient PDX models. These data support clinical evaluation of combined ATR and DNA-PK inhibition in ATM-deficient cancers.

CONSORT diagram of enrolled, randomized, and treated patients in the study. MD, medical doctor; PD, progressive disease; SOC, standard of care. *Registration number was 78, one patient was registered twice.

Clinical outcomes of berzosertib + carboplatin and docetaxel + carboplatin. A and B, Waterfall plots of (A) maximal PSA decline and (B) decline in measurable lesions by RECIST 1.1 from baseline. One patient in arm B (Y, far right) had an unconfirmed PR at cycle 8, followed by progressive disease. C–E, Kaplan–Meier estimates of (C) rPFS, (D) PFS by PCWG3 criteria (“no longer clinically benefiting,” i.e., radiographic or clinical/symptomatic progression), and (E) OS.

HRD status does not demonstrate a statistically significant correlation with clinical benefit in either arm. Kaplan–Meier plots for PFS per PCWG3 criteria with patients across both arms and stratified by (A) HRD status determined by deleterious DDR gene mutations identified by WES of tumor biopsy, (B) RAD51 foci–based IHC, (C) DirectHRD analysis of WGS of cfDNA using a cutoff of 2.5 in the overall cohort, and (D) DirectHRD analysis of WGS of cfDNA using a cutoff of 2.5 in arm B only. NE, not estimable.

A combined ATM-RS biomarker identifies the best responders. A and B, Representative images of ATM (A) and pKAP1 (B) staining by IHC in tumor biopsy samples. The scale bar corresponds to 100 μm. C, Box plot showing pKAP1 levels in samples stratified by ATM status. Horizontal red lines depict the cutoff used to classify samples as ATM-RS–positive or ATM-RS–negative. D and E, Kaplan–Meier plots for PFS in patients across both arms (D) and in Arm B only (E), stratified by the combined ATM-RS classification.

Co-mut plot depicting variants in commonly altered genes in mCRPC (TP53, AR, PIK3CA, PTEN, RB1) and in HRR genes detected in tumor by whole exome sequencing (center triangles, blue) and in cfDNA by targeted panel sequencing at time point 1 (left triangles, purple) and time point 2 (right triangles, green).

Kaplan-Meier curves for PFS with patients stratified by HRD status determined by DirectHRD analysis of WGS data from cfDNA.

Kaplan-Meier curves for PFS stratified by ATM status by immunohistochemistry in tumor biopsy specimens, and scatter plot showing the relationship between PFS and % pKAP1 positivity in tumor biopsy specimens

Representativeness of Study Participants