Huang, R. Stephanie

Abstract Uterine leiomyosarcoma (ULMS) is an orphan disease that frequently recurs and metastasizes, with patients undergoing multiple lines of chemotherapy due to lack of effective therapeutic targets. To address this gap, we used single-cell RNA sequencing and spatial transcriptomic analysis to comprehensively profile ULMS. We uncovered multiple states of tumor cells, including tumor cells with mesenchyme-like features, ischemic tumor cells defined by a MYC program, inflammatory tumor cells with active interferon signaling, and stem cell-like hormone receptor-positive cells. The inferred spatial correlates of these tumor cell states demonstrated unique localization patterns. By correlating these signatures to bulk RNA sequencing data, we demonstrate the relevance of these findings to clinical outcomes. Finally, using the single-cell integration and drug response prediction algorithm (scIDUC), we propose drug predictions that may target specific tumor states. Our findings suggest new avenues for further exploration of individualized and multifaceted therapeutic strategies to treat ULMS.

Abstract Purpose: Germline alterations in homologous recombination repair (gHRR) genes affect the pathogenesis, treatment options, and survival of patients with cancer. However, distinct gHRR gene alterations may differentially affect treatment response and oncogenic signaling. In this study, we interrogated genomic and transcriptomic data and assessed clinical outcomes of patients with gHRR mutations across four BRCA-associated cancers (breast, ovarian, pancreatic, and prostate cancers) to identify therapeutic vulnerabilities. Experimental Design: We assessed 24,309 patients undergoing matched tumor/normal next-generation DNA and RNA sequencing. Annotated gHRR gene variants [germline BRCA1, germline BRCA2, germline PALB2, germline ATM (gATM), and germline CHEK2] were analyzed. HRs were used to assess survival outcomes comparing germline versus sporadic groups. Somatic alterations and their frequencies were compared across gHRR-altered groups. Differential gene expression and gene set enrichment analysis were used to compare transcriptomic profiles. Results: Somatic TP53 mutations were depleted in gATM carriers (P < 0.05) across all four BRCA-associated cancers by up to 2.5-fold. Tumors with germline BRCA1/2 mutations were associated with improved survival in patients with ovarian cancer and had consistent enrichment of TP53 mutations in all four cancers. gATM mutations displayed elevated p53 transcriptional activity in all four cancers, with significance reached in breast and prostate cancers (P < 0.01). In breast, ovarian, and prostate cancers, gATM tumors demonstrated significantly increased inflammatory pathways (P < 0.001). Finally, using gene dependency data, we found that cell lines that were highly dependent on ATM were co-dependent on canonical p53 function. Conclusions: gATM-associated cancers seem to require intact p53 activity and this synthetic essentiality may be used to guide targeted therapies that perturb canonical TP53 function.

e22515 Background: Osteosarcoma is a skeletal malignancy affecting children, adolescents, and young adults. Surgical resection and chemotherapy are the mainstay of therapy with a three-drug regimen - cisplatin, doxorubicin, and methotrexate. However, prognosis remains poor mainly due to chemo-resistance and/or metastases. Emerging evidence has shown that long non-coding RNAs (lncRNAs) are implicated in drug sensitivity and cancer metastasis. Our lab has recently identified strong significant association between cellular sensitivity to cisplatin and doxorubicin and expression of a lncRNA ANRIL in a collection of osteosarcoma cell lines. Methods: SiRNA knockdown experiments were carried out in an osteosarcoma cell line, SAOS2. Cells were exposed to multiple concentrations of cisplatin or doxorubicin. Cellular sensitivity to these two drugs was quantified after siRNA experiment with siRNA targeting ANRIL and scramble control at 24, 48 and 72h. Clinical outcome analysis was performed using data from the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative. Results: We successfully knocked down ANRIL expression in SAOS2 cells with 88.5% efficiency. A significant decreased rate of proliferation was detected for cisplatin (7 uM, 72h) in knockdown cells (16.5% +/- 4.1) compared to scramble control (26.4% +/- 0.8). The cisplatin IC50 was decreased in the siRNA knockdown condition as compared to scramble control at 72h (p = 0.02). For doxorubicin, the IC50 was also significantly decreased in the siRNA knockdown condition as compared to scramble control (p = 0.002 and 0.007 at 24h and 72h, respectively). Survival outcomes, metastases at diagnosis, and percent necrosis (an indicator of treatment response) were evaluated in TARGET osteosarcoma patients (n = 121 and n = 43 for OS, percent necrosis, respectively) with varying degrees of ANRIL expression. Above the median ANRIL expression was associated with a greater number of death (p = 0.004) and higher chances of metastases at diagnosis (p = 0.013). Conclusions: Reducing ANRIL expression led to increased cellular sensitivity to cisplatin and doxorubicin, two key treatment agents for osteosarcoma. Higher ANRIL expression was significantly associated with higher rates of osteosarcoma death and metastases at diagnosis in a clinical cohort of patients. These findings may suggest ANRIL serves as a novel biomarker of cisplatin and doxorubicin sensitivity in osteosarcoma.

Abstract Purpose: Cell-based approaches were used to identify genetic markers predictive of patients' risk for poor response prior to chemotherapy. Experimental Design: We conducted genome-wide association studies (GWAS) to identify single-nucleotide polymorphisms (SNP) associated with cellular sensitivity to carboplatin through their effects on mRNA expression using International HapMap lymphoblastoid cell lines (LCL) and replicated them in additional LCLs. SNPs passing both stages of the cell-based study were tested for association with progression-free survival (PFS) in patients. Phase 1 validation was based on 377 ovarian cancer patients receiving at least four cycles of carboplatin and paclitaxel from the Australian Ovarian Cancer Study (AOCS). Positive associations were then assessed in phase 2 validation analysis of 1,326 patients from the Ovarian Cancer Association Consortium and The Cancer Genome Atlas. Results: In the initial GWAS, 342 SNPs were associated with carboplatin-induced cytotoxicity, of which 18 unique SNPs were retained after assessing their association with gene expression. One SNP (rs1649942) was replicated in an independent LCL set (Bonferroni adjusted P < 0.05). It was found to be significantly associated with decreased PFS in phase 1 AOCS patients (Pper-allele = 2 × 10−2), with a stronger effect in the subset of women with optimally debulked tumors (Pper-allele = 4 × 10−3). rs1649942 was also associated with poorer overall survival in women with optimally debulked tumors (Pper-allele = 9 × 10−3). However, this SNP was not significant in phase 2 validation analysis with patients from numerous cohorts. Conclusion: This study shows the potential of cell-based, genome-wide approaches to identify germline predictors of treatment outcome and highlights the need for extensive validation in patients to assess their clinical effect. Clin Cancer Res; 17(16); 5490–500. ©2011 AACR.