William H. Catherino

Inactivating mutations in TP53 and ATRX are key alterations defining the genomic landscape of uterine leiomyosarcoma (uLMS), creating chronic replication stress and increasing dependency on Ataxia telangiectasia and Rad3-related kinase (ATR) signaling for survival. ATR-CHK1 signaling limits the efficacy of standard-of-care gemcitabine by facilitating the stabilization of stalled replication forks and preventing lethal DNA strand breaks in checkpoint-deficient cells. Here, we show that ATR inhibitors (elimusertib, ceralasertib, berzosertib) preferentially synergize with gemcitabine over docetaxel or doxorubicin, across a panel of TP53-mutant/ATRX-wildtype uLMS models. When simultaneously administered, gemcitabine/elimusertib demonstrated enhanced synergy and efficacy compared to standard-of-care gemcitabine/docetaxel. This combination benefit was absent in a TP53-wildtype uterine sarcoma control line, highlighting TP53 mutation status as a critical predictive biomarker. Combined gemcitabine/ATRi treatment demonstrated reciprocal potency enhancement at low-nanomolar concentrations and significantly increased apoptotic populations compared to monotherapy. Gemcitabine monotherapy induced CHK1 (S345) phosphorylation and a corresponding S-phase arrest. Elimusertib abrogated this checkpoint, transforming protective S-phase arrest into a cytotoxic response characterized by a marked increase in the sub-G1 population. This transition was driven by the inhibition of gemcitabine-induced CHK1 phosphorylation, resulting in marked accumulation of unrepaired DNA damage (γH2AX) and the induction of apoptosis as evidenced by cleaved caspase-3 expression. These findings demonstrate that elimusertib strongly potentiates gemcitabine-induced damage to kill TP53-mutant uLMS in vitro, outperforming standard gemcitabine/docetaxel when concurrently administered. This combination strategy broadens the clinical utility of ATR inhibitors to uLMS harboring the prevalent TP53-mutant/ATRX-wildtype genotype and supports the clinical evaluation of gemcitabine/ATRi combinations as a novel therapy for uLMS.

Objective: Uterine leiomyosarcoma (uLMS) maintains a poor prognosis due to its aggressive behavior and limited therapeutic response. Preclinical progress is severely hindered by traditional subcutaneous models that often fail to metastasize, despite one-third of uLMS patients displaying metastases at diagnosis. This study aimed to establish a reproducible, physiologically accurate orthotopic uLMS murine model that faithfully recapitulates disease progression and chemotherapeutic response.Methods: Primary subcutaneous tumors were established via SK-LMS-1 cell injections in NOD/SCID mice. Harvested xenografts were surgically implanted heterotopically (subcutaneous flank) or orthotopically (uterine myometrium). Eight days post-implantation, mice were randomized to receive intraperitoneal doxorubicin (1.2 mg/kg twice weekly) or vehicle control for three weeks. Tumor growth was tracked longitudinally via MRI (orthotopic) or caliper measurement (subcutaneous). Immunohistochemistry evaluated tumor vitality, microenvironment hypoxia, vascular density, and metastatic spread.Results: Orthotopic tumors exhibited significantly accelerated growth kinetics compared to subcutaneous counterparts. This expansion was supported by co-option of host uterine vasculature, resulting in ample tumor perfusion, suppressed tissue hypoxia, and robust proliferation. Crucially, 37% of primary orthotopic tumors developed spontaneous, multi-organ metastases to pulmonary, omental, and intraperitoneal sites by 4 weeks. Orthotopic tumors demonstrated heightened sensitivity to doxorubicin, achieving a 55% volume reduction compared to 44% in subcutaneous controls, mediated by superior intratumoral drug delivery supported by increased cleaved caspase-3-dependent apoptosis.Conclusions: Surgical orthotopic xenografting recapitulates clinical uLMS, demonstrating site-dependent vascular co-option, spontaneous systemic metastasis, and enhanced chemosensitivity. By mimicking physiological characteristics of human uLMS, this orthotopic model offers significant potential for enhancing understanding of uLMS and evaluating novel therapies.