Helen Zhao

Deintensification Candidate Subgroups in Human Papillomavirus-Related Oropharyngeal Cancer According to Minimal Risk of Distant Metastasis

PURPOSE To define human papillomavirus (HPV) -positive oropharyngeal cancers (OPC) suitable for treatment deintensification according to low risk of distant metastasis (DM). PATIENTS AND METHODS OPC treated with radiotherapy (RT) or chemoradiotherapy (CRT) from 2001 to 2009 were included. Outcomes were compared for HPV-positive versus HPV-negative patients. Univariate and multivariate analyses identified outcome predictors. Recursive partitioning analysis (RPA) stratified the DM risk. RESULTS HPV status was ascertained in 505 (56%) of 899 consecutive OPCs. Median follow-up was 3.9 years. HPV-positive patients (n = 382), compared with HPV-negative patients (n = 123), had higher local (94% v 80%, respectively, at 3 years; P < .01) and regional control (95% v 82%, respectively; P < .01) but similar distant control (DC; 90% v 86%, respectively; P = .53). Multivariate analysis identified that HPV negativity (hazard ratio [HR], 2.9; 95% CI, 2.0 to 5.0), N2b-N3 (HR, 2.9; 95% CI, 1.8 to 4.9), T4 (HR, 1.8; 95% CI, 1.2 to 2.9), and RT alone (HR, 1.8; 95% CI, 1.1 to 2.5) predicted a lower recurrence-free survival (RFS; all P < .01). Smoking pack-years > 10 reduced overall survival (HR, 1.72; 95% CI, 1.1 to 2.7; P = .03) but did not impact RFS (HR, 1.1; 95% CI, 0.7 to 1.9; P = .65). RPA segregated HPV-positive patients into low (T1-3N0-2c; DC, 93%) and high DM risk (N3 or T4; DC, 76%) groups and HPV-negative patients into different low (T1-2N0-2c; DC, 93%) and high DM risk (T3-4N3; DC, 72%) groups. The DC rates for HPV-positive, low-risk N0-2a or less than 10 pack-year N2b patients were similar for RT alone and CRT, but the rate was lower in the N2c subset managed by RT alone (73% v 92% for CRT; P = .02). CONCLUSION HPV-positive T1-3N0-2c patients have a low DM risk, but N2c patients from this group have a reduced DC when treated with RT alone and seem less suited for deintensification strategies that omit chemotherapy.

Chronic Hypoxia Decreases Synthesis of Homologous Recombination Proteins to Offset Chemoresistance and Radioresistance

Hypoxic and/or anoxic tumor cells can have increased rates of mutagenesis and altered DNA repair protein expression. Yet very little is known regarding the functional consequences of any hypoxia-induced changes in the expression of proteins involved in DNA double-strand break repair. We have developed a unique hypoxic model system using H1299 cells expressing an integrated direct repeat green fluorescent protein (DR-GFP) homologous recombination (HR) reporter system to study HR under prolonged chronic hypoxia (up to 72 h under 0.2% O(2)) without bias from altered proliferation, cell cycle checkpoint activation, or severe cell toxicity. We observed decreased expression of HR proteins due to a novel mechanism involving decreased HR protein synthesis. Error-free HR was suppressed 3-fold under 0.2% O(2) as measured by the DR-GFP reporter system. This decrease in functional HR resulted in increased sensitivity to the DNA cross-linking agents mitomycin C and cisplatin but not to the microtubule-interfering agent, paclitaxel. Chronically hypoxic H1299 cells that had decreased functional HR were relatively radiosensitive [oxygen enhancement ratio (OER), 1.37] when compared with acutely hypoxic or anoxic cells (OER, 1.96-2.61). Using CAPAN1 cells isogenic for BRCA2 and siRNA to RAD51, we confirmed that the hypoxia-induced radiosensitivity was due to decreased HR capacity. Persistent down-regulation of HR function by the tumor microenvironment could result in low-fidelity DNA repair and have significant implications for response to therapy and genetic instability in human cancers.

Outcomes of HPV-related oropharyngeal cancer patients treated by radiotherapy alone using altered fractionation.

PURPOSE To report outcome of HPV-related [HPV(+)] oropharyngeal cancer (OPC) managed predominantly by altered-fractionation radiotherapy-alone (RT-alone). METHODS OPCs treated with RT-alone (n = 207) or chemoradiotherapy (CRT) (n = 151) from 2001 to 2008 were included. Overall survival (OS), local (LC), regional (RC) and distant (DC) control were compared for HPV(+) vs. HPV-unrelated [HPV(-)], by RT-alone vs. CRT, and by smoking pack-years (≤ 10 vs. >10). Multivariate analysis identified predictors. RESULTS HPV(+) (n = 277) had better OS (81% vs. 44%), LC (93% vs. 76%), RC (94% vs. 79%) (all p < 0.01) but similar DC (89% vs. 86%, p = 0.87) vs. HPV(-) (n = 81). HPV(+) stage IV CRT (n = 125) had better OS (89% vs. 70%, p < 0.01), but similar LC (93% vs. 90%, p = 0.41), RC (94% vs. 90%, p = 0.31) and DC (90% vs. 83%, p = 0.22) vs. RT-alone (n = 96). Both HPV(+) RT-alone (n = 37) and CRT (n = 67) stage IV minimal smokers had favorable OS (86% vs. 88%, p = 0.45), LC (95% vs. 92%, p = 0.52), RC (97% vs. 93%, p = 0.22), and DC (92% vs. 86%, p = 0.37). RT-alone and heavy-smoking were independent predictors for lower OS but not CSS in multivariate analysis. CONCLUSIONS Overall, HPV(+) RT-alone stage IV demonstrated lower survival but comparable disease control vs. CRT, but no difference was apparent among minimal smokers.

Targeting homologous recombination using imatinib results in enhanced tumor cell chemosensitivity and radiosensitivity

RAD51 is a key protein in the homologous recombination (HR) pathway of DNA double-strand break repair, and HR represents a novel target for cancer therapy. Because imatinib (Gleevec) has been reported to reduce RAD51 protein levels, we tested the clonogenic survival for RT112, H1299, PANC1, and PC3 tumor cell lines of varying p53 status and normal GM05757 normal fibroblasts after exposure to single agent imatinib (0–20 μmol/L; 0–72 hours). We also combined imatinib with DNA damaging agents that are toxic to RAD51-deficient cells, including ionizing radiation, gemcitabine, and mitomycin C. We observed decreased nuclear expression and chromatin binding of RAD51 protein following imatinib treatment. Imatinib also resulted in decreased error-free HR as determined by a flow cytometry–based integrated direct repeat-green fusion protein reporter system; this correlated to reduced RAD51 expression. Clonogenic survival experiments revealed increased cell kill for imatinib-treated cells in combination with ionizing radiation, gemcitabine, and mitomycin C, due in part to mitotic catastrophe. In experiments using imatinib and gemcitabine, tumor cell lines were sensitized to a greater extent than normal fibroblasts. This preservation of the therapeutic ratio was confirmed in vivo using PC3 xenograft growth delay and intestinal crypt cell clonogenic assays. HR inhibition may be an additional mechanism of action for the chemosensitization and radiosensitization of solid tumors with imatinib with preservation of the therapeutic ratio. [Mol Cancer Ther 2009;8(1):203–13]

PTEN Deletion in Prostate Cancer Cells Does Not Associate with Loss of RAD51 Function: Implications for Radiotherapy and Chemotherapy

Purpose: PTEN deletions in prostate cancer are associated with tumor aggression and poor outcome. Recent studies have implicated PTEN as a determinant of homologous recombination (HR) through defective RAD51 function. Similar to BRCA1/2-defective tumor cells, PTEN-null prostate and other cancer cells have been reported to be sensitive to PARP inhibitors (PARPi). To date, no direct comparison between PTEN and RAD51 expression in primary prostate tumors has been reported. Experimental Design: Prostate cancer cell lines and xenografts with known PTEN status (22RV1-PTEN+/+, DU145-PTEN+/−, PC3-PTEN−/−) and H1299 and HCT116 cancer cells were used to evaluate how PTEN loss affects RAD51 expression and PARPi sensitivity. Primary prostate cancers with known PTEN status were analyzed for RAD51 expression. Results: PTEN status is not associated with reduced RAD51 mRNA or protein expression in primary prostate cancers. Decreased PTEN expression did not reduce RAD51 expression or clonogenic survival following PARPi among prostate cancer cells that vary in TP53 and PTEN. PARPi sensitivity instead associated with a defect in MRE11 expression. PTEN-deficient cells had only mild PARPi sensitivity and no loss of HR or RAD51 recruitment. Clonogenic cell survival following a series of DNA damaging agents was variable: PTEN-deficient cells were sensitive to ionizing radiation, mitomycin-C, UV, H2O2, and methyl methanesulfonate but not to cisplatin, camptothecin, or paclitaxel. Conclusions: These data suggest that the relationship between PTEN status and survival following DNA damage is indirect and complex. It is unlikely that PTEN status will be a direct biomarker for HR status or PARPi response in prostate cancer clinical trials. Clin Cancer Res; 18(4); 1015–27. ©2011 AACR.

MRE11 promotes AKT phosphorylation in direct response to DNA double-strand breaks

AKT is hyper-activated in many human cancers and promotes proliferation and cancer cell survival in response to DNA damaging agents. Ionizing radiation (IR) produces DNA double strand breaks (DSB) and activates AKT, however a direct mechanism linking intra-nuclear DSB and AKT signaling is lacking. Here we demonstrate that AKT is phosphorylated following IR in benign and malignant cells and, using colony-forming assays and in vitro rejoining assays, show that AKT promotes non-homologous end joining-mediated DSB repair and cell survival following IR. Further studies revealed that pAKT-S473, but not pAKT-T308 or total AKT, accumulates in the vicinity of IR-induced DSB and co-localizes with γH2AX and ATM-pSer1981. Based on whole-cell IR, nuclear UV microbeam, and endonuclease-induced DSB studies, we observed that pAKT-S473 is up-regulated by a DSB-induced signaling cascade, and this is dependent on the DSB sensor protein, MRE11. MRE11-dependent pAKT-S473 did not require the MRE11 endonuclease domain. The histone ubiquitin ligase RNF168 is also required for DSB-induced pAKT-S473, and DSB-induced pAKT-S473 is independent of DNA-PKcs, PI3K, and ATR. These data demonstrate that DSB activate a signaling cascade that directly promotes a PI3K-independent pathway of AKT phosphorylation that is dependent on MRE11-ATM-RNF168 signaling. Thus, these data directly link the presence of DNA breaks to AKT-mediated cell survival and support AKT as a target for cancer therapy.

Tumor Cell Kill by c-MYC Depletion: Role of MYC-Regulated Genes that Control DNA Double-Strand Break Repair

MYC regulates a myriad of genes controlling cell proliferation, metabolism, differentiation, and apoptosis. MYC also controls the expression of DNA double-strand break (DSB) repair genes and therefore may be a potential target for anticancer therapy to sensitize cancer cells to DNA damage or prevent genetic instability. In this report, we studied whether MYC binds to DSB repair gene promoters and modulates cell survival in response to DNA-damaging agents. Chromatin immunoprecipitation studies showed that MYC associates with several DSB repair gene promoters including Rad51, Rad51B, Rad51C, XRCC2, Rad50, BRCA1, BRCA2, DNA-PKcs, XRCC4, Ku70, and DNA ligase IV. Endogenous MYC protein expression was associated with increased RAD51 and KU70 protein expression of a panel of cancer cell lines of varying histopathology. Induction of MYC in G(0)-G(1) and S-G(2)-M cells resulted in upregulation of Rad51 gene expression. MYC knockdown using small interfering RNA (siRNA) led to decreased RAD51 expression but minimal effects on homologous recombination based on a flow cytometry direct repeat green fluorescent protein assay. siRNA to MYC resulted in tumor cell kill in DU145 and H1299 cell lines in a manner independent of apoptosis. However, MYC-dependent changes in DSB repair protein expression were not sufficient to sensitize cells to mitomycin C or ionizing radiation, two agents selectively toxic to DSB repair-deficient cells. Our results suggest that anti-MYC agents may target cells to prevent genetic instability but would not lead to differential radiosensitization or chemosensitization.

The receptor tyrosine kinase inhibitor amuvatinib (MP470) sensitizes tumor cells to radio- and chemo-therapies in part by inhibiting homologous recombination.

BACKGROUND AND PURPOSE RAD51 is a key protein involved in homologous recombination (HR) and a potential target for radiation- and chemotherapies. Amuvatinib (formerly known as MP470) is a novel receptor tyrosine kinase inhibitor that targets c-KIT and PDGFRα and can sensitize tumor cells to ionizing radiation (IR). Here, we studied amuvatinib mechanism on RAD51 and functional HR. MATERIALS AND METHODS Protein and RNA analyses, direct repeat green fluorescent protein (DR-GFP) assay and polysomal fractioning were used to measure HR efficiency and global translation in amuvatinib-treated H1299 lung carcinoma cells. Synergy of amuvatinib with IR or mitomycin c (MMC) was assessed by clonogenic survival assay. RESULTS Amuvaninib inhibited RAD51 protein expression and HR. This was associated with reduced ribosomal protein S6 phosphorylation and inhibition of global translation. Amuvatinib sensitized cells to IR and MMC, agents that are selectively toxic to HR-deficient cells. CONCLUSIONS Amuvatinib is a promising agent that may be used to decrease tumor cell resistance. Our work suggests that this is associated with decreased RAD51 expression and function and supports the further study of amuvatinib in combination with chemotherapy and radiotherapy.

Abstract 1797: Preclinical evaluation of HM-018, a potent and selective JAK inhibitor in the treatment of myloproliferative disorders

JAK2 kinase mutation V617F is prevalent in myeloproliferative diseases (MPD), including polycythemia vera (PV, 81-99%), essential thrombocytosis (ET, 41-72%) and primary myelofibrosis (PMF, 39-57%). This point mutation constitutively activates the JAK kinase and leads to oncogenic potential of host cells, and thus making JAK2 a promising molecular target for MPD therapy. HM-018 is a small molecule inhibitor against JAK kinase and the compound9s preclinical anti-MPD effects from signal transduction to biological consequences were investigated. HM-018 was found to inhibit JAK kinase 1, 2, 3 and TYK with IC50 of 0.010, 0.006, 0.040 and 0.047 μM, respectively. The compound demonstrated >100 folds selectivity against a panel of 63 kinases. In accordance with enzymatic activity, HM-018 suppressed ligand dependent or constitutive JAK activation in multiple cell lines as evidenced by the decrease of STAT3/5 phosphorylation. As a result, JAK dependent cell proliferation was significantly inhibited by HM-018. EPO-mediated mouse PV model was utilized to evaluate the in vivo efficacy of the compound. HM-018 could shrink enlarged mouse spleen, a typical symptom of PV, in a dose dependent manner accompanied with decreased STAT5 phosphorylation both in animal spleen and in bone marrow after oral dosing for 7 days. To better mimic MPD development in a more clinically-relevant manner, JAK2-V617F-tranfected 32D cells were injected into mice intravenously, and it was observed that oral treatment of HM-018 not only prolonged the animal9s life span, but also reduced MPD-related symptoms, such as spleen weight increase and organ invasion by malignant cells. Meanwhile, HM-018 exhibited a favorable pharmacokinetic profile and acceptable safety window in rats. Based on the preclinical data, HM-018 demonstrated anti-MPD potency both in vitro and in vivo, and the studies have provided rationale to further develop this compound as possible MPD therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1797. doi:1538-7445.AM2012-1797

Abstract 3932: Loss of PTEN sensitizes cells to DNA damaging agents in a RAD51-independent manner

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The PTEN tumor suppressor gene is frequently mutated or deleted in many cancers. While the role of PTEN as an inhibitor of the PI3K/Akt signaling pathway via its intrinsic lipid phosphatase activity has been well-studied, recent data suggest that loss of PTEN contributes to genomic instability through a PI3K/Akt-independent pathway via down-regulation of E2F1-dependent transactivation of Rad51, a key mediator of homologous recombination (HR). Cells with dysfunctional HR are sensitive to inhibitors of Poly(ADP)Ribose Polymerase (PARP) and therefore PTEN loss could sensitize cells to PARP inhibitors in a RAD51-dependent manner. Using PTEN-isogenic cell lines, cells treated with siRNA to PTEN and human prostate cancer cell lines with disparate PTEN status, we assessed whether PTEN loss is associated with altered expression of RAD51 protein. We also evaluated the effects of PTEN loss on sensitivity to PARP inhibition and various DNA damaging agents which sensitize cells based on deficiency in DNA double- and single-strand break repair, nucleotide excision repair and base excision repair (i.e. ionizing radiation (IR), camptothecin (CPT), Mitomycin C, cisplatin, ultraviolet radiation (UV), H2O2 and MMS). We observed that PTEN status did not predict RAD51 expression in PTEN−/− (PC3), PTEN+/− (DU145), and PTEN+/+ (22RV1) prostate cancer cells or xenografts in vitro or in vivo. Using a dedicated tissue microarray of localized prostate cancer, PTEN status (based on FISH) did not predict for RAD51 expression. Furthermore, we observed no difference in RAD51 mRNA or protein expression nor in E2F1 binding to the RAD51 promoter in PTEN-deficient HCT116 colorectal cancer cells or H1299 lung carcinoma cells treated with siRNA to PTEN. PTEN expression also did not alter RAD51 localization to IR-induced nuclear foci or UV microirradiation-induced DNA DSBs. However, using clonogenic assays, we observed that loss of PTEN did miminally sensitize cells to PARP inhibition with increased sensitization to MMC, cisplatin, UV, and IR. PTEN loss did not sensitize tumour cells to MMS, CPT or paclitaxel. Taken together, these data suggest that PTEN influences sensitivity to certain DNA damaging agents in a RAD51-independent manner. To understand this sensitivity, we are documenting gene expression changes in 200 DNA repair and replication genes using NanoString technology. A better understanding of the influence of PTEN on cellular sensitivity to chemo- and radiotherapy may ultimately help to guide treatment options for patients based on PTEN status (Sponsored by CCSRI, PCC and the Terry Fox Foundation). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3932.