Melissa R. Young

A nucleolytic lupus autoantibody is toxic to BRCA2-deficient cancer cells

Cancer cells with defects in DNA repair are highly susceptible to DNA-damaging agents, but delivery of therapeutic agents into cell nuclei can be challenging. A subset of lupus autoantibodies is associated with nucleolytic activity, and some of these antibodies are capable of nuclear penetration. We hypothesized that such antibodies might have potential as therapeutic agents targeted towards DNA repair-deficient malignancies. We identified the lupus autoantibody 5C6 as a cell-penetrating nucleolytic antibody and found that 5C6 has a differential effect on a matched pair of BRCA2-proficient and deficient DLD1 colon cancer cells. 5C6 selectively induced γH2AX in, and suppressed the growth of, the BRCA2-deficient cells. These findings demonstrate the potential utility of 5C6 in targeted therapy for DNA repair-deficient malignancies and strengthen the rationale for studies of additional lupus autoantibodies in order to identify the best candidates for development as therapeutic agents. In addition, the toxic effect of 5C6 on BRCA2-deficient cells provides further support for the hypothesis that some lupus autoantibodies contribute to the lower risk of specific cancers associated with systemic lupus erythematosus.

Adjuvant carboplatin, paclitaxel, and vaginal cuff brachytherapy for stage III endometrial cancer: analysis of outcomes and patterns of recurrence based on pathologic characteristics.

Objective The aim of this study was to evaluate outcomes of patients with stage III endometrial adenocarcinoma treated with surgery followed by adjuvant chemotherapy and vaginal cuff brachytherapy. Methods We retrospectively identified 83 patients treated for 1988 International Federation of Gynecology and Obstetrics (FIGO) stage III endometrial adenocarcinoma at our institution between 2003 and 2010. All patients underwent comprehensive surgical staging. Adjuvant therapy was carboplatin and paclitaxel for 6 cycles and vaginal cuff brachytherapy. For analysis, patients were grouped into type I (FIGO grade 1–2 endometrioid histology, n = 41) or type II (FIGO grade 3, clear cell or papillary serous histology, n = 42) disease. Forty-three patients (52%) had node-positive disease, with similar node-positive rates for type I (n = 21, 51.2%) and type II (n = 22, 52.4%). Results The median follow-up was 38.6 months. There were no isolated vaginal failures. The estimated 3-year disease-free survival (DFS) and overall survival (OS) for type I versus type II were 92.4% versus 58.0% (P = 0.001) and 97.2% versus 65.8% (P = 0.002), respectively. The 3-year DFS and OS for node negative versus node positive were 85.0% versus 63.6% (P = 0.02) and 84.2% versus 78.0% (P = 0.02), respectively. Associations between type I histology and node-negative disease with improved DFS and OS persisted on multivariate analysis. Conclusions Our institutional approach of adjuvant chemotherapy and vaginal cuff brachytherapy for stage III endometrial cancer seemed acceptable for patients with low-risk histology or node-negative disease. In contrast, higher rates of failure among those with high-risk histology and/or node-positive disease support intensification of therapy in these subsets.

Optimizing a Lupus Autoantibody for Targeted Cancer Therapy

The specificity of binding by antibodies to target antigens is a compelling advantage to antibody-based cancer therapy, but most antibodies cannot penetrate cells to affect intracellular processes. Select lupus autoantibodies penetrate into cell nuclei, and the potential for application of these antibodies in cancer therapy is an emerging concept. Here, we show that a divalent lupus anti-DNA autoantibody fragment with enhancing mutations that increase its ability to penetrate cell nuclei and bind DNA causes accumulation of DNA double-strand breaks in and is highly and selectively toxic to cancer cells and tumors with defective homology-directed repair of DNA double-strand breaks. These findings provide proof of principle for the use of optimized lupus autoantibodies in targeted cancer therapy.

Paxillin and embryonic PolyAdenylation Binding Protein (ePABP) engage to regulate androgen-dependent Xenopus laevis oocyte maturation - A model of kinase-dependent regulation of protein expression

Steroid-triggered Xenopus laevis oocyte maturation is an elegant physiologic model of nongenomic steroid signaling, as it proceeds completely independent of transcription. We previously demonstrated that androgens are the main physiologic stimulator of oocyte maturation in Xenopus oocytes, and that the adaptor protein paxillin plays a crucial role in mediating this process through a positive feedback loop in which paxillin first enhances Mos protein translation, ensued by Erk2 activation and Erk-dependent phosphorylation of paxillin on serine residues. Phosphoserine-paxillin then further augments Mos protein translation and downstream Erk2 activation, resulting in meiotic progression. We hypothesized that paxillin enhances Mos translation by interacting with embryonic PolyAdenylation Binding Protein (ePABP) on polyadenylated Mos mRNA. Knockdown of ePABP phenocopied paxillin knockdown, with reduced Mos protein expression, Erk2 and Cdk1 activation, as well as oocyte maturation. In both Xenopus oocytes and mammalian cells (HEK-293), paxillin and ePABP constitutively interacted. Testosterone (Xenopus) or EGF (HEK-293) augmented ePABP-paxillin binding, as well as ePABP binding to Mos mRNA (Xenopus), in an Erk-dependent fashion. Thus, ePABP and paxillin work together in an Erk-dependent fashion to enhance Mos protein translation and promote oocyte maturation.

Impact of vaginal cylinder diameter on outcomes following brachytherapy for early stage endometrial cancer

Objective To examine the outcomes (tolerability, toxicity, and recurrence) of vaginal brachytherapy (VBT) among endometrial cancer (EC) patients treated with small cylinder size. Methods Patients with EC who received adjuvant VBT between September 2011 and December 2015 were reviewed. Patients were fitted with the largest vaginal cylinder they could comfortably accommodate, from 2.0–3.0 cm diameter. Small cylinders were defined as size 2.3 cm or less. Patient, tumor, or treatment characteristics were correlated with need for small cylinders. Treatment tolerability, measures of gastrointestinal (GI), genitourinary (GU), and vaginal toxicity, and rates of recurrence were analyzed. Results Three hundred four patients were included. Small cylinders were used in 51 patients (17%). Normal body mass index (BMI; p<0.001), nulligravidity (p<0.001), and shorter vaginal length (p<0.001) were associated with small cylinder size. There was no acute or late grade 3 toxicity. Rates of acute (grade 1–2) GI, GU, or vaginal symptoms were low (10%, 11%, and 19%, respectively). Small cylinder size was associated with increased likelihood of reporting acute GI (p<0.05) but not GU or vaginal symptoms. Small cylinder size was associated with higher risk of grade 1–2 vaginal stenosis (odds ratio [OR]=4.7; 95% confidence interval [CI]=1.5–14.7; p=0.007). There was no association between cylinder size and recurrence rate (p=0.55). Conclusion VBT is generally very well tolerated, however, patients fitted with smaller cylinders (commonly nulligravid and low BMI) may have increased side effects. Further study to improve the dosimetry of VBT for patients requiring small cylinders may be worthwhile.

Who benefits from chemoradiation in stage III–IVA endometrial cancer? An analysis of the National Cancer Data Base

OBJECTIVE Adjuvant therapy for advanced endometrial cancer (AEC) is not standardized. We investigated whether regional radiotherapy with chemotherapy (CRT) compared to chemotherapy alone (CT) was associated with improved overall survival (OS) in an AEC cohort and among subgroups by stage and histologic grade. METHODS Women who received CT or CRT after hysterectomy and bilateral salpingo-oophorectomy for FIGO stage III-IVA AEC diagnosed in 2004-2012 were identified in the National Cancer Data Base. Multilevel modeling was used to identify covariates associated with treatment selection. OS was compared using Kaplan-Meier estimates, the log-rank test, Cox proportional hazards regression, and propensity score matching. RESULTS We identified 9837 patients, of whom 6358 (65%) received CT and 3479 (35%) received CRT. Median follow-up was 59.6months. OS was higher in patients receiving CRT compared to CT (70% v 55% at 5years, log-rank P<0.001). Controlling for stage, histologic grade, tumor size, age, comorbidity and race, CRT remained independently associated with improved OS (HR 0.63, 95% CI 0.57-0.70, P<0.001). When stratified by stage and histologic grade, there was a significant OS benefit for stage IIIA, IIIB, IIIC, grade 2, and grade 3 (all P<0.001), a trend for stage IVA (P=0.06), but no benefit for grade 1 (P=0.91). On multivariable subgroup analyses, these findings persisted, including lack of benefit in grade 1 patients (HR 0.72, P=0.14). These results were further confirmed after propensity score matching. CONCLUSIONS Adjuvant CRT for AEC was associated with improved OS, except for patients with well-differentiated disease, who fared equally well with CT alone.

Adjuvant Therapy Use and Survival in Stage II Endometrial Cancer

Objective Radiotherapy (RT) is an established adjuvant treatment for stage II endometrioid endometrial carcinoma (EEC). The role of chemotherapy (CT) in stage II EEC is less proven. We used the National Cancer Data Base to identify factors associated with adjuvant CT in stage II EEC and to explore whether receipt of CT was associated with improved overall survival (OS). Methods/Materials Women diagnosed in 2010 to 2013 with International Federation of Obstetrics and Gynecology stage II EEC (grades 1–3) after hysterectomy and bilateral salpingo-oophorectomy were identified in the National Cancer Data Base. Multivariable logistic regression was used to identify covariates associated with receipt of CT. Overall survival among patients receiving RT, CT, or chemoradiotherapy (CRT) after surgery was compared using Kaplan-Meier estimates, the log-rank test, Cox proportional hazards regression, and propensity score matching. Results We identified 6102 stage II EEC patients. There were 358 patients (6%) who received adjuvant CT alone and 525 (9%) who received CRT; the remainder received RT alone (n = 1906; 31%) or no adjuvant treatment (n = 3313; 54%). The presence of lymphovascular invasion (odds ratio, 3.58; P < 0.001) and grade 3 disease (odds ratio, 3.40; P < 0.001) was strongly associated with receipt of CT or CRT. The OS at 3 years for the entire cohort was 89%. On multivariable analysis, CT versus RT was associated with worse OS (hazard ratio [HR], 2.12 [95% confidence interval, 1.46–3.06]; P < 0.001), whereas CRT versus RT was not associated with improved OS (HR, 1.07 [95% confidence interval, 0.71–1.62]; P = 0.781). After propensity score matching, there remained no difference in OS between RT and CRT (HR, 1.14; P = 0.614). Conclusions Patients with stage II EEC have an excellent prognosis, and most undergo observation or receive adjuvant RT in the United States. Receipt of CT (alone or with RT) was not associated with an OS advantage compared with RT alone in this observational cohort. Randomized trials will help clarify the role of CT in stage II patients.

Chapter 45 – Intensity Modulated Radiotherapy and Image Guidance

External beam radiotherapy is the most common nonsurgical approach to the treatment of locally advanced prostate cancer. Over the last several decades, significant advances in radiation treatment delivery, planning, and image guidance have resulted in the ability to significantly reduce radiation exposure to normal tissues while simultaneously allowing for increased radiation dose delivery to the prostate in men with localized prostate cancer. This has resulted in improved cure rates with a reduction in toxicity of treatment. The ability to deliver more conformal radiation to higher doses to the prostate is made possible with the advent of intensity-modulated radiation paired with image guidance techniques such as fiducial markers, cone beam CT, and implanted radiofrequency transponders. The evolution of such technologies and a review of the resulting disease control and toxicity rates are described.

Abstract 654: Targeting K-ras mutant cancer cells with a lupus anti-guanosine antibody

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Aberrant G-protein signaling due to mutations in genes encoding the Ras family of small GTPases is associated with approximately 30% of human cancers, and K-ras is one of the most commonly mutated Ras proteins. Development of targeted therapies that are preferentially toxic to cancer cells harboring K-ras mutations is therefore an important goal in cancer research. We have identified an unusual cell-penetrating lupus autoantibody, 4H2, as a potential candidate for development as a targeted therapy for tumors with activating K-ras mutations. 4H2 is an anti-guanosine antibody that has previously been shown to penetrate living cells and inhibit cAMP formation. Taken together, the ability of 4H2 to bind guanosine (a component of GDP/GTP) and to inhibit cAMP formation suggests that 4H2 perturbs G-protein signaling. We therefore hypothesized that 4H2 would be selectively toxic to cancer cells with aberrant G-protein signaling due to activating K-ras mutations compared to cells without mutations in K-ras. To test this hypothesis, we evaluated the effects of 4H2 on a panel of isogenic pairs of cells with and without activating K-ras mutations, including Cal12T lung cancer cells (with and without a G12C activating K-ras mutation), NCI-H1975 lung cancer cells (with and without a G12D activating K-ras mutation), and SW48 colon cancer cells (with and without a G12A activating K-ras mutation). Immunofluorescence experiments confirmed that 4H2 penetrates into and localizes to the cytoplasm of cells with and without an activating K-ras mutation. In cell proliferation assays the matched pairs of cells were treated with 4H2 while growing as subconfluent monolayers, and 4H2 was observed to significantly inhibit the proliferation of the cells with activating K-ras mutations but not the cells without K-ras mutations. Next, colony formation assays confirmed that treatment with 4H2 was significantly more toxic to cells with activating K-ras mutations compared to cells without K-ras mutations. Mechanistically, Western blot analyses suggest a differential effect of 4H2 on the MAP kinase and Akt signaling pathways in K-ras mutant and wild type cells, consistent with the hypothesis that 4H2 interferes with G-protein associated intracellular signaling pathways. In summary, this work has demonstrated that 4H2 is an intriguing candidate for further study and development as a potential targeted therapy for tumors with activating mutations in K-ras. Citation Format: Melissa R. Young, Philip W. Noble, Richard H. Weisbart, James E. Hansen. Targeting K-ras mutant cancer cells with a lupus anti-guanosine antibody. [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 654. doi:10.1158/1538-7445.AM2014-654

Abstract 4220: A cell-penetrating nucleolytic lupus autoantibody damages DNA and is toxic to BRCA2-deficient cancer cells

Identification of therapeutic agents that are more toxic to malignant cells than normal cells is a key goal in cancer research. Many tumor cells harbor genetic defects that distinguish them from normal cells, and some of these defects have the potential to be exploited in the development of targeted therapies for cancer. Cancer cells with defects in DNA repair are highly susceptible to DNA-damaging agents, but delivery of therapeutic agents into cell nuclei can be challenging. A subset of lupus autoantibodies is associated with nucleolytic activity, and some of these antibodies are capable of nuclear penetration. We hypothesized that such antibodies might be capable of damaging DNA in cell nuclei and therefore have potential as therapeutic agents targeted towards DNA repair-deficient malignancies, such as tumors that are BRCA2-deficient. To test this hypothesis we screened a panel of cell-penetrating lupus autoantibodies for nucleolytic activity, and we identified 5C6 as an antibody of interest. 5C6 catalyzes degradation of single and double-stranded DNA in vitro and induces phospho-H2AX formation in BRCA2-deficient cells. When tested on a matched pair of BRCA2-proficient and deficient cancer cells, 5C6 selectively suppressed the growth of and was significantly more toxic to the BRCA2-deficient cells. These findings demonstrate the potential utility of 5C6 in targeted therapy for DNA repair-deficient malignancies and strengthen the rationale for studies of additional lupus autoantibodies in order to identify the best candidates for development as therapeutic agents. In addition, the toxicity of 5C6 for BRCA2-deficient cells provides further support for the hypothesis that some lupus autoantibodies contribute to the unusual cancer risk profile associated with systemic lupus erythematosus. Citation Format: Philip W. Noble, Melissa R. Young, Richard H. Weisbart, James E. Hansen. A cell-penetrating nucleolytic lupus autoantibody damages DNA and is toxic to BRCA2-deficient cancer cells. [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 4220. doi:10.1158/1538-7445.AM2014-4220