Yu, Jia

Abstract Purpose: We hypothesized that integrated analysis of cancer types from different lineages would reveal novel molecularly defined subgroups with unique therapeutic vulnerabilities. On the basis of the molecular similarities between subgroups of breast and ovarian cancers, we analyzed these cancers as a single cohort to test our hypothesis. Experimental Design: Identification of transcriptional subgroups of cancers and drug sensitivity analyses were performed using mined data. Cell line sensitivity to Hsp90 inhibitors (Hsp90i) was tested in vitro. The ability of a transcriptional signature to predict Hsp90i sensitivity was validated using cell lines, and cell line- and patient-derived xenograft (PDX) models. Mechanisms of Hsp90i sensitivity were uncovered using immunoblot and RNAi. Results: Transcriptomic analyses of breast and ovarian cancer cell lines uncovered two mixed subgroups comprised primarily of triple-negative breast and multiple ovarian cancer subtypes. Drug sensitivity analyses revealed that cells of one mixed subgroup are significantly more sensitive to Hsp90i compared with cells from all other cancer lineages evaluated. A gene expression classifier was generated that predicted Hsp90i sensitivity in vitro, and in cell line- and PDXs. Cells from the Hsp90i-sensitive subgroup underwent apoptosis mediated by Hsp90i-induced upregulation of the proapoptotic proteins Bim and PUMA. Conclusions: Our findings identify Hsp90i as a potential therapeutic strategy for a transcriptionally defined subgroup of ovarian and breast cancers. This study demonstrates that gene expression profiles may be useful to identify therapeutic vulnerabilities in tumor types with limited targetable genetic alterations, and to identify molecularly definable cancer subgroups that transcend lineage.

Abstract INTRODUCTION: Breast cancer is the most common invasive cancer and the second leading cause of cancer death in women. Approximately 20% of breast cancer tumors overexpress human epidermal growth factor receptor 2 (HER2), which is associated with an aggressive clinical phenotype and with a poorer prognosis. The development of trastuzumab, an immunoglobulin G monoclonal antibody directed against HER2, has changed the treatment paradigm for this disease. However, approximately 15-20% still does not respond to anti-HER2 therapy. Furthermore, despite advances in the management of metastatic HER2-positive breast cancer, response rate varies greatly, with most patients eventually succumbing to their disease. It is crucial to develop additional therapeutic strategies to improve outcomes for HER2 positive patients, especially those with tumors that do not respond to anti-HER2 therapy. METHODS and RESULTS: Using HER2+ breast cancer cell lines, we found that an USP7 inhibitor, P5091, dramatically sensitized cancer cells to HER2 inhibitor, trastuzumab treatment. To confirm it in vivo, we tested the drug combination in BT474 xenografts and found P5091 and trastuzumab synergistically suppressed tumor growth. Furthermore, using trastuzumab resistant HER2+ breast cancer cell line, we found that USP7 inhibitor, P5091, can dramatically re-sensitize trastuzumab-resistant BT474 cell line to trastuzumab treatment both in vitro and in vivo. More importantly, using patient derived xenograft (PDX) HER2+ models, we confirmed the synergistic combination effect of P5091 and trastuzumab. Mechanistically, USP7 inhibitor, P5091 led to significantly increased degradation of all four HER family members (HER1-4). When we knocked down USP7 by RNA interference or small hairpin RNA (shRNA), we observed similar substantial decrease in HER family member proteins. CONCLUSIONS: We have demonstrated the feasibility of using USP7 inhibitor P5091 to sensitize HER2+ breast cancers to trastuzumab treatment. These results provide a rationale for the use of combination therapy of USP7 inhibitors and HER2 inhibitors to treat HER2+ breast cancer. This combination therapy might also be effective in other cancers with upregulated HER signaling, such as GI cancer, lung cancer or ovarian cancer. Citation Format: Jia Yu, Bo Qin, Judy Boughey, Matthew Goetz, Liewei Wang. The role of USP7 and USP7 inhibitor in HER2+ breast cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2980.

Purpose The majority of patients with cancer receive treatments that are minimally informed by omics data. We propose a precision medicine computational framework, PANOPLY (Precision Cancer Genomic Report: Single Sample Inventory), to identify and prioritize drug targets and cancer therapy regimens. Materials and Methods The PANOPLY approach integrates clinical data with germline and somatic features obtained from multiomics platforms and applies machine learning and network analysis approaches in the context of the individual patient and matched controls. The PANOPLY workflow uses the following four steps: selection of matched controls to the patient of interest; identification of patient-specific genomic events; identification of suitable drugs using the driver-gene network and random forest analyses; and provision of an integrated multiomics case report of the patient with prioritization of anticancer drugs. Results The PANOPLY workflow can be executed on a stand-alone virtual machine and is also available for download as an R package. We applied the method to an institutional breast cancer neoadjuvant chemotherapy study that collected clinical and genomic data as well as patient-derived xenografts to investigate the prioritization offered by PANOPLY. In a chemotherapy-resistant patient-derived xenograft model, we found that that the prioritized drug, olaparib, was more effective than placebo in treating the tumor ( P < .05). We also applied PANOPLY to in-house and publicly accessible multiomics tumor data sets with therapeutic response or survival data available. Conclusion PANOPLY shows promise as a means to prioritize drugs on the basis of clinical and multiomics data for an individual patient with cancer. Additional studies are needed to confirm this approach.

Abstract Triple-negative breast cancer (TNBC) is characterized by elevated locoregional recurrence risk despite aggressive local therapies. New tumor-specific radiosensitizers are needed. We hypothesized that the ATR inhibitor, VX-970 (now known as M6620), would preferentially radiosensitize TNBC. Noncancerous breast epithelial and TNBC cell lines were investigated in clonogenic survival, cell cycle, and DNA damage signaling and repair assays. In addition, patient-derived xenograft (PDX) models generated prospectively as part of a neoadjuvant chemotherapy study from either baseline tumor biopsies or surgical specimens with chemoresistant residual disease were assessed for sensitivity to fractionated radiotherapy, VX-970, or the combination. To explore potential response biomarkers, exome sequencing was assessed for germline and/or somatic alterations in homologous recombination (HR) genes and other alterations associated with ATR inhibitor sensitivity. VX-970 preferentially inhibited ATR-Chk1-CDC25a signaling, abrogated the radiotherapy-induced G2–M checkpoint, delayed resolution of DNA double-strand breaks, and reduced colony formation after radiotherapy in TNBC cells relative to normal-like breast epithelial cells. In vivo, VX-970 did not exhibit significant single-agent activity at the dose administered even in the context of genomic alterations predictive of ATR inhibitor responsiveness, but significantly sensitized TNBC PDXs to radiotherapy. Exome sequencing and functional testing demonstrated that combination therapy was effective in both HR-proficient and -deficient models. PDXs established from patients with chemoresistant TNBC were also highly radiosensitized. In conclusion, VX-970 is a tumor-specific radiosensitizer for TNBC. Patients with residual TNBC after neoadjuvant chemotherapy, a subset at particularly high risk of relapse, may be ideally suited for this treatment intensification strategy. Mol Cancer Ther; 17(11); 2462–72. ©2018 AACR.

The testis‐specific Y‐encoded‐like protein (TSPYL) gene family includes TSPYL1 to TSPYL6. We previously reported that TSPYL5 regulates cytochrome P450 (CYP) 19A1 expression. Here we show that TSPYLs, especially TSPYL 1, 2, and 4, can regulate the expression of many CYP genes, including CYP17A1, a key enzyme in androgen biosynthesis, and CYP3A4, an enzyme that catalyzes the metabolism of abiraterone, a CYP17 inhibitor. Furthermore, a common TSPYL1 single nucleotide polymorphism (SNP), rs3828743 (G/A) (Pro62Ser), abolishes TSPYL1's ability to suppress CYP3A4 expression, resulting in reduced abiraterone concentrations and increased cell proliferation. Data from a prospective clinical trial of 87 metastatic castration‐resistant prostate cancer patients treated with abiraterone acetate/prednisone showed that the variant SNP genotype (A) was significantly associated with worse response and progression‐free survival. In summary, TSPYL genes are novel CYP gene transcription regulators, and genetic alteration within these genes significantly influences response to drug therapy through transcriptional regulation of CYP450 genes.

Abstract Elucidating mechanisms of chemoresistance is critical to improve cancer therapy, especially for the treatment of pancreatic ductal adenocarcinoma (PDAC). Genome-wide association studies have suggested the less studied gene HEAT repeat-containing protein 1 (HEATR1) as a possible determinant of cellular sensitivity to different chemotherapeutic drugs. In this study, we assessed this hypothesized link in PDAC, where HEATR1 expression is downregulated significantly. HEATR1 silencing in PDAC cells increased resistance to gemcitabine and other chemotherapeutics, where this effect was associated with increased AKT kinase phosphorylation at the Thr308 regulatory site. Mechanistically, HEATR1 enhanced cell responsiveness to gemcitabine by acting as a scaffold to facilitate interactions between AKT and the protein phosphatase PP2A, thereby promoting Thr308 dephosphorylation. Consistent with these findings, treatment with the AKT inhibitor triciribine sensitized HEATR1-depleted PDAC cells to gemcitabine, suggesting that this therapeutic combination may overcome gemcitabine resistance in patients with low HEATR1 expression. Clinically, we found that HEATR1 downregulation in PDAC patients was associated with increased AKT phosphorylation, poor response to tumor resection plus gemcitabine standard-of-care treatment, and shorter overall survival. Collectively, our findings establish HEATR1 as a novel regulator of AKT and a candidate predictive and prognostic indicator of drug responsiveness and outcome in PDAC patients. Cancer Res; 76(3); 572–81. ©2015 AACR.

Abstract Background Cancer genomic investigations have identified recurrent genomic aberrations critical for cancer initiation, progression, and metastases. However, these investigations are typically performed in isolation, and the effects of treatment on the clonal selection of tumor cells are mostly unknown. We hypothesized that molecular profiling of residual tumors after neoadjuvant chemotherapy (NAC) would identify new drug targets/pathways in patients at high risk for disease recurrence. To better identify clonal populations of resistant breast cancer cells, we utilized DNA content-based flow sorting of nuclei to identify and isolate clonal populations for aCGH and next generation sequencing (NGS) both before and after NAC. Methods The Breast Cancer Genome Guided Therapy Study (BEAUTY) (NCT 02022202) is a prospective study of patients with high-risk breast cancer treated with neoadjuvant 12 weekly paclitaxel (T) +/- trastuzumab followed by 4 cycles of anthracycline based chemotherapy. Tumor tissue from baseline, residual disease from surgery, distant metastases, and patient derived xenografts (PDX) are obtained for cell sorting by DNA ploidy, aCGH, RNA and exome sequencing. Results: 140 patients have been enrolled, 104 have completed surgery and 30 unique PDX have been established corresponding to 26 patients prior to chemotherapy and 4 from residual disease at surgery. Baseline exome and RNA sequencing is complete in 140. Currently, genomic analyses of flow sorted matched baseline, surgical, PDX, and distant disease samples are available in 6 patients. Substantial genomic variation was observed in the surgical sample compared to the primary tumor including gain of oncogenic drivers (EGFR) and loss of negative regulators (ATG5) (Table). The PDX recapitulated these events with excellent fidelity compared to the corresponding human tumor. In patients with TNBC, RNA seq obained from matched samples demonstrated changes in immune related pathways. Evaluation of drug targets/pathways identified in the resistant tumors are ongoing using the PDX and sequencing of the remaining matched baseline/surgical disease will be reported. Clonal changes occurring over time in patients with residual disease or disease recurrence after NACTumor SubtypeResidual Cancer BurdenDisease StatusClonal Aberration Changes (baseline and post NAC)TNBC (AR subtype)3Contralateral lymph node recurrence at 150 days2p25.2 - p25.1 amplicon lost at recurrence; 6p21.32 -p21.31 amplicon lost at recurrenceTNBC (BL 1)3Bone/liver/lymph node recurrence at 135 days5q11.2 amplicon gained at surgery; 12p13.33 - p13.2 amplicon gained at surgeryTNBC (BL 1)3Disease-free at day 1506q21 amplicon lost at sugeryTNBC (BL 2)0Brain recurrence at 390 daysChr 2 chromothripsis at surgeryLuminal B3Progression during chemotherapy7p12.1 - p11.2 amplicon gained at surgeryLuminal HER23Disease-free at day 3609q33.2 amplicon lost at surgery; 15q13.1 - q13.3 amplicon lost at surgery; 15q22.2 - q24.1 amplicon lost at surgery Conclusions: We observed substantial evolutionary changes in residual breast tumors remaining after NAC. Our findings suggest that a comprehensive assessment of the mutational landscape that has evolved during NAC can inform drug development in high risk breast cancer patients. Citation Format: Matthew P Goetz, Michael T Barrett, Krishna R Kalari, Vera J Suman, Sarah A McLaughlin, Alvaro Moreno-Aspitia, Ann M Moyer, Donald W Northfelt, Richard J Gray, Jason Sinnwell, Douglas Mahoney, Poulami Barman, Peter Vedell, Xiaojia Tang, Kevin Thompson, Travis Dockter, Katie Jones, Sara J Felten, Amy Conners, Jeanette Eckel-Passow, Hughes Sicotte, Steven N Hart, Jia Yu, Daniel W Visscher, Eric D Wieben, Cloann Schultz, Minetta C Liu, James N Ingle, Liewei Wang, Richard W Weinshilboum, Judy C Boughey. Impact of neoadjuvant chemotherapy on the clonal composition of breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr PD3-3.

Abstract INTRODUCTION Patient derived xenografts (PDX) may better reflect individual patient (pt) tumor biology; however, the feasibility of collecting PDX from percutaneous tumor biopsies (PTB) in the neoadjuvant setting is unknown. Furthermore, drug response phenotypes observed in PDX have not been prospectively compared to the corresponding pt clinical outcomes. METHODS The Breast Cancer Genome Guided Therapy Study (BEAUTY) is a prospective Mayo study of pts with high-risk breast cancer treated with neoadjuvant weekly paclitaxel (T) +/- trastuzumab followed by anthracycline based chemotherapy. PTB (at baseline) and residual surgical tissue (after all chemotherapy) are obtained for next generation sequencing (NGS) and PDX. Tumor biopsies (1-2 cores from 14 gauge needle) were implanted with Matrigel &lt;1 hour of collection in the flanks of NOD-SCID or NSG mice. Low dose estradiol was supplemented in the drinking water. Primary outgrowth rate was defined as PDX tumor volume &gt;50 mm3. Take rate was defined as development of at least 1 stably transplantable xenograft line/pt. To determine whether clinical T response assessed by MRI corresponded with in vivo T response, pretreatment PDX from 5 pts were injected into NOD-SCID mice (20 mice per pt PDX) and when tumors reached 100-200mm3, mice were randomized to no treatment vs T (20 mg/kg, ip. every 3-4 days). Two of these 5 patients had a MRI response defined as &gt;30% decrease in longest lesion. RESULTS Pretreatment PTB from 81 unique pts were implanted in 251 mice (2-4 mice/pt). PDX outgrowth rates were 33.3% (27/81 pts) and 22 stable PDX were established (overall take rate 27.2%). Take rates were as follows: triple negative breast cancer (46%; 13/28); HER2 (27%; 6/22), Luminal B (13%; 3/22), and luminal A (0%; 0/9). Residual surgical tumor (after all treatment) from 17 pts was injected into 85 mice (average 5 mice/pt) and the initial outgrowth rate was 23% (4/17) with 3 stably transplantable lines established. PDX, derived from pretreatment PTB of 5 pts (2 responders and 3 non-responders), were assessed for in vivo T response. The size of the T treated group was significantly smaller than the no treatment group for the PDX derived from the 2 clinical responders, with complete disappearance of tumor by 18 days. In contrast, the PDX derived from the 3 clinical non-responders had no evidence for T response. CONCLUSIONS We have demonstrated the feasibility of using PTB to establish PDX in a prospective neoadjuvant clinical study and have demonstrated similar T drug response phenotypes in in the PDX as seen in the corresponding pt. These data suggest that PDX generated prospectively may be useful for biomarker validation and the development and individualization of new drug therapy. Funded by the Mayo Clinic Center for Individualized Medicine and the Mayo Clinic Cancer Center Citation Format: Jia Yu, Ping Yin, Bowen Gao, Jason P. Sinnwell, Ann M. Moyer, Daniel W. Visscher, Amy L. Conners, Travis J. Dockter, Krishna R. Kalari, Xiaojia Tang, Kevin J. Thompson, Hugues Sicotte, Douglas W. Mahoney, Steven N. Hart, Peter T. Vedell, Poulami Barman, Katie N. Jones, Sarah A. McLaughlin, John A. Copland, Alvaro Moreno Aspitia, Donald W. Northfelt, Richard J. Gray, Vera J. Suman, Jeanette E. Eckel Passow, Eric D. Wieben, James N. Ingle, Zhenkun Lou, Gianrico Farrugia, Richard Weinshilboum, Matthew P. Goetz, Judy C. Boughey, Liewei Wang. Feasibility of using percutaneous tumor biopsies from a prospective neoadjuvant breast cancer study to develop patient derived xenografts and assess in vivo chemotherapy sensitivity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1195. doi:10.1158/1538-7445.AM2014-1195