Hou, Xiaonan

Abstract Poly (ADP) ribose polymerase inhibitors (PARPi) have shown promising activity against recurrent ovarian cancers, particularly in women with germ line mutations of BRCA1/2. The efficacy of PARP inhibitors can, however, be decreased by acquired drug resistance. Autophagy or “self-eating” is one mechanism implicated in cancer progression and therapy resistance. Autophagy is a catabolic mechanism that can degrade organelles and long-lived proteins to provide energy for cancer cells under nutrient poor conditions or in the presence of stress. The energy provided from autophagy is used to fuel the DNA repair process and metabolic needs in cancer cells. Autophagy can protect cancer cells from chemotherapy or can enhance the response to certain drugs. In this study, we have asked whether autophagy contributes to the resistance of ovarian cancer cells to PARPi. Our group has found that four PARP inhibitors (olaparib, niraparib, rucaparib and talazorapib) induce autophagy in 8 ovarian cancer cell lines, detected by LC3A to LC3B conversion on western blot analysis and punctate GFP-LC3 fluorescence. Transmission electron microscopy confirmed the increased autophagosome formation in olaparib treated cells. To test whether autophagy contributes to the resistance of OC cells to olaparib, we determined the impact of autophagy inhibition on the sensitivity of OC cell lines to olaparib by either using chloroquine, hydroxychloroquine or LYS05 to block hydrolysis of proteins and lipids in autophagosomes or using siRNA against ATG5 or ATG7 to prevent formation of autophagosome. Enhancement of PARPi activity was observed in 6 out 8 ovarian cancer cell lines. Moreover, a combination of olaparib and chloroquine proved more effective than either single agent in a xenograft model of the OVCAR8 ovarian cancer cell line and in a patient derived xenograft model. Mechanistically, olaparib decreased PARP activity leading to DNA damage and accumulation of γ-H2AX. Olaparib treatment increased reactive oxygen species (ROS) and phosphorylation of ATM, while decreasing the phosphorylation of AKT and mTOR. We also observed an increase in PTEN, a well-recognized negative regulator of AKT. Inhibition of PTEN decreases LC3 conversion, suggesting PTEN as an important regulator of olaparib-induced autophagy in ovarian cancer cells. Taken together, our data demonstrate for the first time that olaparib induces autophagy in ovarian cancer cells and provides an adaptive mechanism of resistance to PARPi. Olaparib-induced autophagy may depend upon activation of ATM and PTEN by ROS, downregulating pAKT/pmTOR signaling. Moreover we have identify unique combinations of therapy with PARPi and CQ or other inhibitors of autophagy that could improve outcomes for ovarian cancer patients. Citation Format: Janice M. Santiago-O'Farrill, Saravut J. Weroha, Xiaonan Hou, Lan Pang, Philip Rask, Zhen Lu, Robert C. Bast. PARP inhibitor-induced autophagy provides an adaptive mechanism of drug resistance in preclinical models of ovarian cancer [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 4768.

e17039 Background: Treatment strategies are needed for platinum-resistant recurrent ovarian cancer. Glucocorticoid receptor (GR) is associated with poor prognosis in ovarian cancer. GR signaling inhibits chemotherapy-induced cell death via transcriptional regulation of anti-apoptotic proteins. Non-selective (mifepristone) and selective GR antagonists (Relacorilant, Corcept Therapeutics) are under clinical development (eg. NCT02046421, NCT03776812). We hypothesized that GR antagonism would improve chemotherapy sensitivity in ovarian cancer patient-derived xenograft (PDX) models. Methods: Five PDX models were selected based on GR expression above median RNA seq levels. Tissues were transplanted into 300 female SCID-Bg mice at the Mayo Clinic. Once tumor diameter reached 0.2 cm, mice were randomized into treatment arms (2-25 mice per arm) and treated intraperitoneal (i.p): control (9% saline, CTL) on day 0 (D0), 1, 7, 8, 14, 15, 21, 22; mifepristone (MIF), 15 mg/kg D0, 1, 7, 8, 14, 15, 21, 22; relacorilant (134), 20 mg/kg D0, 1, 7, 8, 14, 15, 21, 22; carboplatin (CBDCA) at the MTD, 51 mg/kg D1, 8, 15, 22; CBDCA + MIF; CBDCA + 134. Tumor area was measured by ultrasound weekly (D0-D28), and the ratio calculated relative to D0. Tumor growth curves were compared between arms for each PDX via a linear mixed effects regression test of coincident curves. Time to progression was also evaluated and will be presented. Results: In 1/5 models (PH160), CBDCA + MIF significantly suppressed tumor growth compared to CBDCA alone (p < 0.05, Table 1). Addition of 134 to CBDCA did not suppress tumor growth compared to CBDCA alone in any model. In the 2 models with no difference of CBDCA versus CTL (PH160, PH454), addition of a GR antagonist to CBDCA suppressed tumor growth versus CTL. Conclusions: Addition of GR antagonists can improve response to chemotherapy. Work is ongoing to identify the GR transcriptional network, tumor, and host characteristics which predict responses to GR antagonists. [Table: see text]

e17038 Background: Carcinosarcomas (CS) are aggressive gynecologic malignancies defined by a biphasic tumor histology of carcinomatous and sarcomatous components. High expression of glucocorticoid receptor (GR) is associated with aggressive tumor biology in triple-negative breast, ovarian, and endometrial cancers. GR activation results in transcriptional regulation of epithelial-mesenchymal transition (EMT) genes. Therefore, we hypothesized that the sarcomatous component of Müllerian CS may be associated with relatively higher GR expression compared to the carcinomatous portion. Methods: Müllerian CSs from the University of Chicago tissue bank diagnosed from 5/19/2009 – 8/25/2014 were identified and pathology confirmed (RL). In addition, we evaluated GR expression in a mouse PDX CS model. Immunohistochemical expression of nuclear estrogen (ER), progesterone (PR), androgen (AR), and GR was quantified using the H-score. Scores between the sarcomatous and carcinomatous components were compared using a paired t-test. Results: Mean GR H-score was relatively higher in the sarcomatous vs carcinomatous component, overall (144.44 vs 38.89, P = 0.0014). Mean H-score also differed between sarcomatous and carcinomatous components for ER (3.06 vs 63.06, P = 0.016), PR (1.67 vs 47.22, P = 0.004), and AR (0 vs 2.78, P = 0.056). The significantly higher sarcomatous GR expression was replicated in IHC evaluation of the mouse PDX model. Conclusions: GR expression is significantly higher in the sarcomatous than the carcinomatous component of Müllerian CS, while ER, PR, and AR expression is significantly lower. Recent data suggest that an EMT gene signature is more prominently expressed in the sarcomatous versus carcinomatous component. We hypothesize that GR expression and activity may mediate EMT gene expression and represent a therapeutic target for CS. [Table: see text]

Abstract In lung adenocarcinoma, gene fusion involving the anaplastic lymphoma kinase (ALK) gene leads to overexpression of its tyrosine kinase domain, which serves as the oncogenic driver in lung tumors carrying such a molecular alteration. Similar fusions involving ROS1, RET and NTRK are also found in lung cancer and tumors with oncogenic fusions often respond well to targeted therapy. However, these tumors do develop resistance to the targeting drug and patients eventually relapse while on treatment. Although newer drugs have been developed, it remains a challenge to effectively control cancer recurrence after targeted therapy. In vivo tumor models carrying these fusions, particularly those that have progressed while on targeted treatment, will help to facilitate further drug evaluations prior to their administration to the patient as well as offer an opportunity to better understand the molecular mechanisms driving disease onset, progression, and drug resistance in tumors carrying the fusion of interest. Here, we describe the first patient-derived tumor xenograft (PDX) carrying an STRN-ALK fusion. The patient was diagnosed with stage IV lung adenocarcinoma in 2011. She was initially treated with crizotinib and remained stable until 2015. At the time of disease progression, a biopsy was taken with patient consent under CT guidance from a chest wall mass using an 18-gauge needle and then implanted into two SCID-beige mice subcutaneously and into the fat pad of the kidney. Palpable tumor was observed in both mice at about 28 weeks post implantation and passaged at 36 weeks. They have been passaged two successive times and viably frozen. Pathologic analysis of the PDX showed that both tumors have the same mucinous morphology as observed in the original biopsy sample. RNA-seq and matepair analysis revealed that the tumors carry a rare fusion involving the first three exons of STRN and exon 20 to 3’ end of the ALK gene. The patient has been on ceritinib since January 2016 and remains stable as of last follow up. Several unique features contributed to the success of this study. 1) The metastatic chest wall lesion used for the PDX developed was easily accessible on the chest wall using a relative large 18-gauge needle for CT-guided biopsies. 2) The slow growth of the tumor biopsy mirrors the clinical course of the disease observed in the patient. 3) The patient remains clinically stable and continues to respond to TKi. The availability of a viable PDX tumor model for this rare STRN-ALK fusion offers a unique opportunity to identify the most appropriate drug for the patient at the time of disease progression as well as the opportunity to examine the unique molecular features associated with this rare ALK fusion. This work was supported in part by the National Foundation for Cancer Research Hillsberg Lung Cancer Translational Research Grant and by the Biomarker Discovery Program at the Mayo Clinic Center for Individualized Medicine. Citation Format: HongZheng Res, XiaoNan Hou, Karlyn E. Pierson, Patric Eiken, Asha Nair, PingYang Yang, Joanne Eunhee Yi, Aaron S. Mansfield, Jin Jen. Establishment of a unique patient-derived tumor model positive for STRN-ALK fusion from a patient with stage IV lung adenocarcinoma [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr A17.

Ovarian cancer is the most lethal gynecologic malignancy. About 75% of ovarian cancer patients relapse and/or develop chemo‐resistant disease after initial response to standard‐of‐care treatment with platinum‐based therapies. HER2 amplifications and overexpression in ovarian cancer are reported to vary, and responses to HER2 inhibitors have been poor. Next generation sequencing technologies in conjunction with testing using patient‐derived xenografts (PDX) allow validation of personalized treatments. Using a whole‐genome mate‐pair next generation sequencing (MPseq) protocol, we identified several high grade serous ovarian cancers (HGS‐OC) with DNA alterations in genes encoding members of the ERBB2 pathway. The efficiency of anti‐HER2 therapy was tested in three different PDX lines with the identified alterations and high levels of HER2 protein expression. Treatment responses to pertuzumab or pertuzumab/trastuzumab were compared in each PDX line WITH standard carboplatin and paclitaxel combination treatment. In all three PDX models, HER2‐targeted therapy resulted in significant inhibition of tumor growth compared with untreated controls. However, the responses in each case were inferior to those to chemotherapy, even for chemo‐resistant lines. When chemotherapy and HER2‐targeted therapy were administered together, a significant regression of tumor was observed after 6 weeks of treatment compared with chemotherapy alone. Post‐treatment analysis of these tissues revealed that inhibition of the ERBB2 pathway occurred at the level of phosphorylation and expression of downstream targets. In conclusion, while targeting of presumably activated ERBB2 pathway alone in HGS‐OC results in a modest treatment benefit, a combination therapy including both chemotherapy drugs and HER2 inhibitors provides a far better response. Further studies are needed to address development of recurrence and sensitivity of recurrent disease to HER2‐targeted therapy.

Abstract Background: Acquired PARP inhibitor (PARPi) resistance in high-grade serous ovarian cancer (HGSOC) as a result of restored homologous recombination has been observed following secondary mutations that restore full-length protein in BRCA1, BRCA2, RAD51C, and RAD51D. Additionally, loss of BRCA1 methylation has also been shown to confer resistance. However, little is known about the role of RAD51C methylation in acquired PARPi resistance. In ARIEL2 Part 1, a phase 2 study of the PARPi rucaparib in ovarian carcinoma, four (2%) tumors demonstrated RAD51C methylation. The present study utilizes HGSOC patient derived xenografts (PDXs) and recurrent samples from ARIEL2 to assess the role of RAD51C methylation in the development of PARPi resistance. Methods: To drive PARPi resistance, PDX039, an extremely PARPi-sensitive model lacking demonstrable mutations in DNA repair genes, was treated cyclically with niraparib (100 mg/kg) for 21 days, after which the tumor was allowed to regrow and re-established in new mice for the next treatment round. To evaluate the frequency of methylation change, RAD51C methylation was analyzed in 12 rucaparib-treated mice (300 or 450 mg/kg) harboring PDX183, a PARPi-sensitive model without mutations in DNA repair genes. Global changes in gene expression following development of PARPi resistance were assessed by RNA sequencing. RAD51C promoter methylation was evaluated by bisulfite sequencing. Subsequent functional analysis included qRT-PCR, IHC, and western blot. DNA damage response pathways are being evaluated by immunofluorescence ex vivo following niraparib, rucaparib, or IR. Results: PDX039 grew through PARPi treatment by the third and fourth cycle of therapy. RAD51C was the only DNA repair gene to show significant change in RNAseq analysis (log2 fold-change=8.43; p=2e-192), corresponding with a loss of RAD51C methylation. Moreover, after just one round of PARPi treatment, RAD51C methylation was lost in 1 of 12 PARPi-treated PDX183 xenografts. RAD51C methylation loss ultimately resulted in restoration of expression, for which functional analysis is ongoing. Analysis of patient samples is currently underway. Conclusions: In HGSOC PDX models, RAD51C methylation affords PARPi sensitivity in the absence of DNA repair gene mutations. Treatment pressure with PARPi can reverse RAD51C methylation and restore RAD51C expression. Isolated changes in methylation of the RAD51C locus are sufficient to restore HR and convey PARPi resistance. Citation Format: Rachel M. Hurley, Ksenija Nesic, Cordelia McGehee, Olga Kondrashova, Maria I. Harrell, Paula A. Schneider, Xiaonan Hou, Cristina Correia, Karen S. Flatten, Giada V. Zapparoli, Alexander Dobrovic, Kevin K. Lin, Thomas C. Harding, Andrea E. Wahner Hendrickson, Elizabeth M. Swisher, Matthew Wakefield, S. John Weroha, Clare L. Scott, Scott H. Kaufmann. Loss of RAD51C promoter hypermethylation confers PARP inhibitor resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5885.

Abstract Ovarian cancer (OC) is most lethal gynecologic malignancy. About 75% of OC patients relapse and/or develop chemo-resistant disease after initial response to standard-of-care treatment with platinum-based therapies. HER2 amplifications and overexpression in OC are reported to vary and responses to HER2 inhibitors have been feeble. Next generation sequencing technologies in conjunction with testing using patient-derived xenografts (PDX) allow validation of personalized treatments. Using whole-genome mate-pair next generation sequencing protocol we profiled a set of serous OC to identify copy number changes and gene fusions. Specific focus was on DNA alterations which were therapeutically targetable. The efficiency of anti-Her2 therapy was tested in three different PDX lines with alterations at genes of ERBB pathway and various levels of HER2 protein expression. Tumor in one line harbored a duplication of ERBB2 and high expression of HER2 protein. Second PDX model had a fusion at NRG1 gene, a ligand for HER4 and HER4 receptors, that was highly expressed on protein level. Third line harbored an amplification of NRG3 gene, a known ligand for HER4 receptor. Treatment responses to pertzumab/trastuzumab were compared in each PDX line to standard carboplatin and paclitaxel combination treatment. In all three PDX models HER2-targeted therapy resulted in significant inhibition of tumor growth compared to untreated control. However, the responses in each were inferior to chemotherapy, even in chemo-resistant case. There was an extra benefit when chemotherapy and HER-2 targeted therapy were administered together, as a significant regression of tumor was observed after 6 weeks of treatment using this regiment compared to chemotherapy alone. Post-treatment analysis of the tissues, nevertheless, revealed inhibition of ERBB2 pathway on the level of phosphorylation and expression of downstream targets. In conclusion, targeting of presumably activated ERBB2 pathway alone in OC results in a modest treatment benefit. A combination therapy including both chemo drugs and Her2 inhibitors provides far better response. Further studies are needed to address development of recurrence and sensitivity of recurrent disease to Her2 targeted therapy. Citation Format: Irina V. Kovtun, Zhang Piyan, Faye R. Harris, Xiaonan Hou, Saravut J. Weroha, George Vasmatzis. Targeting ERBB2 pathway in ovarian cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-A19.