Project 1: Summary/Abstract Leiomyosarcoma (LMS) is one of the most common sarcomas and is a considerable therapeutic challenge because 50% of patients develop metastases for which current chemotherapy provides clinical benefit to only a small minority. We have shown that most LMS have chromosomal instability (CIN), which results in part from defects in homologous recombination and from ubiquitous TP53 and RB1 inactivation. Our recent studies show that these CIN aberrations are associated with hyper-dependency on DNA protein kinase (DNA-PK), which – along with ATM and ATR – is a member of the PI3K-like kinase family of DNA damage response (DDR) enzymes. DNA-PK is a key repair mechanism for double-strand DNA damage, thereby maintaining CIN below genotoxic thresholds. In Project 1, we wish to develop therapies that maximize intrinsic LMS genotoxic stress, particularly be targeting DNA-PK and by discovering more selective ways to target the DNA-PK pathway. To this end, Aim 1 determines whether relevant biomarkers (biochemical DNA-PK pathway activation, DNA damage response, and genomic evidence of CIN) are found homogeneously within a metastasis and in different metastases from a given patient. Aim 1 also determines whether partially vs. completely inactivating TP53 mutations have differing impact on LMS CIN and DNA-PK activation levels and therefore differing predictive relevance with respect to therapies targeting DNA-PK and DNA integrity. By determining if assays of DNA-PK activation and DNA damage are effectively demonstrated by LMS IHC and genomic profiles, Aim 1 shows whether these assays can be used as robust and reproducible correlative science assessments for our subsequent (Aim 3b) clinical trial combining low-dose doxorubicin and DNA-PK inhibition. Aim 2 provides biologic insights needed to achieve efficacy and selectivity for DNA-PK inhibition and other DDR-targeted therapies in LMS. These in vitro studies include DDR CRISPR-i and CRISPR-a screens, which are performed with/without DNA-PKi, PARPi and doxorubicin treatment. The CRISPR screens aim to discover DNA-PK inhibitor synthetic lethals and resistance mechanisms, and the biologic insights from these screens are enhanced by performing RNAseq and phosphoproteome profiles in the same LMS conditions, and by characterizing LMS cells with acquired resistance to DNA-PKi or doxorubicin. Aim 3 is the clinical translation for Project 1, which encompasses in vitro, and murine preclinical validations and an initial clinical trial of doxorubicin coupled with DNA-PK inhibition. Here we test the hypothesis that a very low dose of doxorubicin sensitizes LMS cells to DNA-PKi, thereby converting CIN into a liability while minimizing nonneoplastic cell toxicity.