Lymphoid malignancies are a group of highly heterogeneous diseases frequently associated with constitutive activation of the nuclear factor kappa B (NF-κB) signaling pathway. Parthenolide is a natural compound used to treat migraines and arthritis and found to act as a potent NF-κB signaling inhibitor. This study evaluated in vitro parthenolide efficacy in lymphoid neoplasms. We assessed parthenolide metabolic activity in NCI-H929 (MM), Farage (GCB-DLBCL), Raji (BL), 697 and KOPN-8 (B-ALL), and CEM and MOLT-4 (T-ALL), by resazurin assay. Cell death, cell cycle, mitochondrial membrane potential (ΔΨ_mit), reactive oxygen species (ROS) and reduced glutathione (GSH) levels, activated caspase-3, FAS-ligand, and phosphorylated NF-κB p65 were evaluated using flow cytometry. CMYC, TP53, GPX1, and TXRND1 expression levels were assessed using qPCR. Our results showed that parthenolide promoted a metabolic activity decrease in all cell lines in a time-, dose-, and cell-line-dependent manner. The mechanism induced by parthenolide was demonstrated to be cell line dependent. Nonetheless, parthenolide promoted cell death by apoptosis with significant ROS increase (peroxides and superoxide anion) and GSH decrease combined with a ΔΨ_mit reduction across all studied cell lines. Despite the need to further understand parthenolide mechanisms, parthenolide should be considered as a possible new therapeutic approach for B- and T-lymphoid malignancies.

Purpose: Mutations in the ATM gene are common in multiple cancers, but clinical studies of therapies targeting ATM-aberrant cancers have yielded mixed results. Refinement of ATM loss of function (LOF) as a predictive biomarker of response is urgently needed. Experimental Design: We present the first disclosure and preclinical development of a novel, selective ATR inhibitor, ART0380, and test its antitumor activity in multiple preclinical cancer models. To refine ATM LOF as a predictive biomarker, we performed a comprehensive pan-cancer analysis of ATM variants in patient tumors and then assessed the ATM variant-to-protein relationship. Finally, we assessed a novel ATM LOF biomarker approach in retrospective clinical data sets of patients treated with platinum-based chemotherapy or ATR inhibition. Results: ART0380 had potent, selective antitumor activity in a range of preclinical cancer models with differing degrees of ATM LOF. Pan-cancer analysis identified 10,609 ATM variants in 8,587 patient tumors. Cancer lineage–specific differences were seen in the prevalence of deleterious (Tier 1) versus unknown/benign (Tier 2) variants, selective pressure for loss of heterozygosity, and concordance between a deleterious variant and ATM loss of protein (LOP). A novel ATM LOF biomarker approach that accounts for variant classification, relationship to ATM LOP, and tissue-specific penetrance significantly enriched for patients who benefited from platinum-based chemotherapy or ATR inhibition. Conclusions: These data help to better define ATM LOF across tumor types in order to optimize patient selection and improve molecularly targeted therapeutic approaches for patients with ATM LOF cancers.

Purpose: DNA polymerase theta (Polθ) is the central component of DNA double-strand break repair through microhomology-mediated end joining (MMEJ). Targeting Polθ for cancer therapy has been proposed due to its tumor-selective expression, as well as its role in the survival of homologous recombination–deficient (HRD) cancer cells. Additionally, inhibition of Polθ has been shown to potentiate the effects of DNA damage, such as that caused by poly (ADP-ribose) polymerase (PARP) inhibition. In this study, we present the pharmacologic profile of ART6043, a potent, selective, allosteric, small-molecule inhibitor of the polymerase function of Polθ. Experimental Design: A series of in vitro studies were performed to demonstrate the biochemical and cellular on-target activity of ART6043 in relevant model systems, focusing on combination with PARP inhibitors. Subsequent in vivo studies established a pharmacokinetic/pharmacodynamic/efficacy relationship and explored ART6043’s ability to potentiate the efficacy of PARP inhibitors across a range of cell line-derived xenograft (CDX), patient-derived xenograft (PDX), and genetically engineered mouse models of HRD tumors. Results: ART6043 inhibits Polθ with low nmol/L potency and specifically abrogates MMEJ in cells. Additionally, ART6043 inhibits the survival of HRD cells and potentiates the efficacy of PARP inhibitors in vitro. Furthermore, ART6043 has excellent pharmaceutical properties, is well tolerated, and induces tumor regressions in combination with PARP inhibitors in vivo in HRD models. Conclusions: The data demonstrate strong potential for ART6043 as an anticancer therapeutic for HRD tumors in combination with PARP inhibitors. Based on this, combination therapy of ART6043 with PARP inhibitor treatment is being tested in an ongoing phase I/IIa clinical trial (NCT05898399).

Inhibition of DNA polymerase theta (Polθ), an essential enzyme for repairing DNA double-strand breaks (DSBs) via microhomology-mediated end joining (MMEJ), has proven to be an exquisitely effective monotherapy in HR-deficient tumor models. In addition, Polθ inhibition (Polθi) can induce tumor-selective radiosensitization, but unlike its monotherapy use, no clinically actionable biomarkers have yet been identified to predict this effect. Here, we profiled 54 cancer cell lines and found that Polθi induces substantial radiosensitization in most models, although with marked variability not explained by indicators of Polθ activity. To pinpoint molecular determinants of radiosensitization by Polθi, we performed a CRISPR knockout screen which revealed loss of the TP53BP1/Shieldin pathway component SHLD2 ( FAM35A ) as a vulnerability to Polθi combined with RT. We found that SHLD2 is deleted in a subset of human prostate cancers, frequently alongside PTEN loss, an adverse prognostic factor. We demonstrated that SHLD2 loss not only increases sensitivity to RT alone, as reported previously, but also enhances the radiosensitizing effect of Polθi, independently of PTEN status and without requiring HR deficiency. Moreover, our findings support a model in which SHLD2 deficiency increases Polθ dependence following RT, with Polθ activity limiting DSB accumulation and chromosomal instability, via a compensatory mechanism independent of canonical MRE11/CtIP-mediated DNA end resection. In summary, we found that SHLD2 loss is a collateral vulnerability that can be exploited through combined treatment with Polθi and RT.