Kaufmann, Scott H

Abstract Background: At the time of ovarian cancer platinum resistance, standard therapies have limited activity. New anti-cancer drug combinations may lead to improved clinical outcomes. Activity of topoisomerase I (Top I) inhibitors or poly(ADP-ribose) polymerase (PARP) inhibitors have been reported in ovarian cancer and have demonstrated potentiation in other models. Objective: To investigate the efficacy of the Top I inhibitor etirinotecan pegol (NKTR-102) and the PARP inhibitor rucaparib (CO-338) as single agents and in combination in patient-derived xenograft (PDX) models of ovarian cancer. Methods: Five ovarian cancer PDX models in SCID mice were treated for 28 days with NKTR-102 (10 mg/kg/q7d IV), rucaparib (150 mg/kg/qd PO), or combination NKTR-102 plus rucaparib at the same doses and schedules. Of the five PDX models, four were platinum resistant and one was a platinum-sensitive positive control. Each treatment arm had a total of 10 mice and animals were observed daily for body weight and general condition. Tumor size was assessed weekly by ultrasound. The primary endpoint was change in tumor cross-sectional area (cm2) from pretreatment baseline to final tumor burden. Western blot was performed to investigate potential biomarkers of response. Results: Compared to vehicle-treated controls, statistically significant improvement (p<0.05) was observed in 3 of 5 models in single agent rucaparib treated tumors and in 5 of 5 models in single agent NKTR-102 treated tumors. The combination of NKTR-102 plus rucaparib also demonstrated statistically significant improvement compared to vehicle-treated controls in 5 of 5 models. The combination of NKTR-102 plus rucaparib induced maximal tumor regression in 2 of 5 tumors with a trend towards smaller tumors compared to single agent treatment arms. This was not found to be statistically significant in the 28 day study period under which the experiment was performed. Exploratory analysis to assess candidate drug response determinants (e.g.Top I, PAPR I, and PARP polymer formation levels) was investigated. Conclusion: NKTR-102 and rucaparib are well tolerated in mice and demonstrate activity in platinum-resistant and platinum-sensitive ovarian cancer PDX models. Combination therapy demonstrated a trend towards decreased tumor size when compared to single agent activity during treatment. Current efforts are focused on extending the time on study for the PDX models evaluated to determine if statistical superiority of the NKTR-102/rucaparib combination can be established. Citation Format: Caroline C. Nitschmann, Rachel M. Hurley, Marc A. Becker, Xiaonan Hou, Ute Hoch, Thomas C. Harding, Deborah H. Charych, Scott H. Kaufmann, Paul Haluska, Saravut J. Weroha. Antineoplastic activity of Top I inhibitor etirinotecan pegol (NKTR-102) and PARP inhibitor rucaparib (CO-388) in platinum-resistant high-grade serous BRCA WT ovarian cancer PDX models. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B50.

Abstract Background: Defects in homologous recombination (HR) DNA repair are common in high grade serous ovarian, peritoneal and fallopian tube carcinoma (HGSOC). Other than mutations in BRCA1 and BRCA2 (BRCA1/2), the molecular causes of HR deficiency have not been well delineated. The frequency of HR deficiency in non-serous ovarian carcinoma is not known. We previously identified that germline and somatic HR mutations in 13 genes in OC was associated with improved survival. We sought to replicate these data in an independent tumor set, while sequencing a larger number of DNA repair genes. Methods: We used BROCA-HR to sequence 65 DNA repair genes in germline (blood) and tumor DNA from 363 women with ovarian (262), peritoneal (91), or fallopian tube (10) carcinoma from an institutional tissue bank. Cox proportional hazards models, adjusted for stage and residual tumor after cytoreduction, were used to assess associations between mutations in DNA repair genes in HR or related pathways and overall survival from diagnosis. Results: In total, 96/363 (25%) patients had 59 germline and 45 somatic deleterious mutations in HR genes, including 34 germline and 22 somatic BRCA1/2 mutations. Damaging mutations found in other HR genes included 25 germline and 23 somatic in the following genes: 5 ATM, 6 ATR, 1 BAP1, 2 BARD1, 6 BLM, 2 BRIP1, 2 BRE, 1 BRCC3, 2 CHEK2, 1 ERCC1, 1 FANCG, 1 FANCI, 2 FANCL, 8 FANCM, 1 NBN, 2 PALB2, 1 RAD51D, 1 RBBP8, 1 SLX4, 1 UIMC1 and 1 XRCC2. Of 322 HGSOC, HR mutations occurred in 86 patients (26.7%) including 54 (16.8%) germline and 32 somatic (9.9%). HR mutation rates were similar across the different non-serous histologies. Of 13 clear cell OC, 2 (15.3%) had germline mutations in non-BRCA1/2 HR genes and 3 (23.1%) had PTEN/PIK3CA mutations. Of 27 endometrioid OC, 3 (11.1%) had HR mutations (1 germline BRCA1, and 2 non-BRCA1/2 somatic HR mutations). PTEN/PIK3CA mutations were identified in 9 (2.8%) HGSOC, 3 (23.1%) clear cell and 2 (7.5%) endometrioid. HR mutations demonstrated a trend towards improved long-term overall survival (hazard ratio = 0.78, 96% CI 0.57-1.05) after accounting for stage and debulking status. Conclusions: These data not only indicate that damaging HR mutations, both germline and somatic, are found in endometrioid and clear cell as well as HGSOC, but also confirm our previous results showing an association between HR mutations and improved overall survival in OC. We are now sequencing 163 additional non-serous OC to augment the current analysis and these data will be available at presentation. Citation Format: Maria I. Harrell, Matthew J. Maurer, Ethan P. Heinzen, Ming K. Lee, Kimberly R. Kalli, Kathy J. Agnew, Ann L. Oberg, Tom Walsh, Barbara M. Norquist, Lynn C. Hartmann, Scott H. Kaufmann, Elizabeth M. Swisher. Homologous recombination mutations and overall survival in high-grade serous, endometrioid, and clear cell ovarian carcinomas. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A07.

A peptide-universal nucleotide conjugate that mimics the topoisomerase I-DNA covalent complex was synthesized and then utilized to raise monoclonal antibodies.

Abstract Purpose: APOBEC3 DNA cytosine deaminase family members normally defend against viruses and transposons. However, deregulated APOBEC3 activity causes mutations in cancer. Because of broad expression profiles and varying mixtures of normal and cancer cells in tumors, including immune cell infiltration, it is difficult to determine where different APOBEC3s are expressed. Here, we ask whether correlations exist between APOBEC3 expression and T-cell infiltration in high-grade serous ovarian cancer (HGSOC), and assess whether these correlations have prognostic value. Experimental Design: Transcripts for APOBEC3G, APOBEC3B, and the T-cell markers, CD3D, CD4, CD8A, GZMB, PRF1, and RNF128 were quantified by RT-qPCR for a cohort of 354 HGSOC patients. Expression values were correlated with each other and clinical parameters. Two additional cohorts were used to extend HGSOC clinical results. Immunoimaging was used to colocalize APOBEC3G and the T-cell marker CD3. TCGA data extended expression analyses to additional cancer types. Results: A surprising positive correlation was found for expression of APOBEC3G and several T cell genes in HGSOC. Immunohistochemistry and immunofluorescent imaging showed protein colocalization in tumor-infiltrating T lymphocytes. High APOBEC3G expression correlated with improved outcomes in multiple HGSOC cohorts. TCGA data analyses revealed that expression of APOBEC3D and APOBEC3H also correlates with CD3D across multiple cancer types. Conclusions: Our results identify APOBEC3G as a new candidate biomarker for tumor-infiltrating T lymphocytes and favorable prognoses for HGSOC. Our data also highlight the complexity of the tumor environment with respect to differential APOBEC family gene expression in both tumor and surrounding normal cell types. Clin Cancer Res; 22(18); 4746–55. ©2016 AACR.

Abstract Background: Cytarabine and an anthracycline, the mainstay of AML therapy, affords a five-year survival below 30%. Accordingly, there is a continuing need for improved AML therapies. CPX-351 encapsulates within liposomes a fixed 5:1 molar ratio of cytarabine and daunorubicin that is synergistic (in vitro) and markedly improved in anti-leukemic activity (in vivo). Promising evidence of improved remission and survival rates with CPX-351 compared to standard of care treatments has also been observed clinically and a pivotal Phase 3 trial is currently ongoing. Because Chk1 inhibition has been shown to enhance the actions of both cytarabine and anthracyclines, there has been interest in assessing the effect of CPX-351 when combined with Chk1 inhibitors. Here we describe the evaluation of CPX-351 alone and in combination with the Chk1 inhibitors MK-8776 and LY2603618 in AML cell lines and in clinical AML samples ex vivo. Methods: Effects of CPX-351 on Chk1 phosphorylation and cell cycle distribution in the human AML lines U937 and HL-60 were analyzed by immunoblotting and flow cytometry. Effects of Chk1 inhibitors on CPX-351-induced apoptosis were assessed by flow cytometry after propidium iodide staining (DNA cleavage), Hoechst staining (nuclear fragmentation) and colony formation in soft agar (long-term survival). Effects CPX-351 ± MK-8776 on clinical AML samples were assayed using clonogenic assays. Results: Treatment of U937 cells with CPX-351 resulted in phosphorylation of Chk1 on Ser317 and Ser296 (sites of ATR-mediated phosphorylation and autophosphorylation, respectively) as well as S phase slowing consistent with Chk1 activation. Co-treatment of AML cell lines with Chk1 inhibitors + CPX-351 resulted in increased apoptotic morphological changes and DNA fragmentation as well as diminished clonogenic survival compared to CPX-351 alone. The CPX-351/MK-8776 combination displayed enhanced antiproliferative effects against some clinical AML isolates ex vivo compared to CPX-351 alone, with additional assays ongoing. Conclusions: Collectively, these results show that Chk1 inhibition enhances the cytotoxicity of CPX-351 in AML cell lines and some clinical AML samples, offering the possibility that CPX-351 may be well suited for combining with such molecularly targeted agents. Citation Format: Nicole D. Vincelette, Larry M. Karnitz, Judith E. Karp, Douglas B. Smith, Allan D. Hess, Lawrence D. Mayer, Scott H. Kaufmann. CPX-351 (cytarabine:daunorubicin liposome for injection) anti-leukemia activity is potentiated by Chk1 inhibition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3479. doi:10.1158/1538-7445.AM2015-3479

Significance There is a need for a robust and efficient eukaryotic polypeptide display platform for the discovery and affinity maturation of antibodies and other scaffolds, offering a powerful addition to the existing display technologies that use microorganisms. This study is significant in that it demonstrates just such a technology, avian leukosis virus (ALV) polypeptide display, supplying an innovative and substantive departure from the status quo of antibody display that uses platforms based on microorganisms. The use of eukaryotic cells provides the assurance of maximizing the proper folding, posttranslational modifications, and display of the single-chain Fv antibodies and the virus fusion proteins in virions and on the cell surface. ALV display offers virus and cell display platforms in a eukaryotic expression system.

Abstract Background: PARP inhibitors are thought to be effective therapy for cancers with homologous recombination (HR) deficiency, best shown to date in patients (pts) with a germline BRCA (gBRCA) mutation. The Cancer Genome Atlas (TCGA) estimated ∼ 50% of high-grade serous ovarian carcinoma (OC) pts have an HR defect (HRD) due to a BRCA mutation (germline or somatic), other HR gene mutation, or another mechanism, e.g., BRCA1 promoter methylation. A randomized maintenance trial of olaparib in recurrent OC demonstrated a clinical benefit in both gBRCA-mutated and gBRCA-wild-type pts. Currently, the best molecular predictors of PARPi response (other than a BRCA mutation) are not known. Platinum sensitivity, often used as a surrogate predictive indicator for PARPi response, is inadequate. A better technique, such as molecular analysis of tumor tissue, could be a superior method for selection of pts for PARPi therapy. Methods: The rucaparib development plan in OC employs a unique strategy of conducting the key trials in parallel, allowing the Phase 2 (ARIEL2) to complete first so that a molecular HRD signature, associated with sub-groups most likely to benefit, can be prospectively applied to the final analysis of the Phase 3 pivotal trial (ARIEL3). ARIEL2 is an ongoing, single-arm, open-label biomarker study designed to refine the molecular signature associated with a response to rucaparib. Eligible pts (n=180) have relapsed, platinum-sensitive, high-grade OC and measurable disease. All pts undergo a pre-dose biopsy and provide archival tumor tissue. Tumor tissue HR status is assessed using Foundation Medicine's next generation sequencing (NGS) platform and University of Washington's BROCA-HR panel, with the current HRD algorithm developed using in vitro/in vivo and TCGA (and similar) bioinformatic data. The key efficacy analyses to be correlated with tumor HR status are response by RECIST v1.1, GCIG-defined CA-125 response, and PFS. The number of gBRCA pts is limited to maximize non-gBRCA response predictors. Exploratory biomarkers include assessment of tissue NHEJ competency (candidate predictive biomarker) and circulating tumor DNA (candidate efficacy surrogate). The optimized algorithm will then be tested prospectively in ARIEL3 (n=540), an international, randomized (2:1), double-blind, placebo-controlled maintenance trial in platinum-sensitive OC in remission after platinum-based therapy. The primary endpoint of ARIEL3 is PFS in molecularly-defined HRD subgroups as determined by NGS analysis of archival tumor tissue using the optimized algorithm, refined (as required) through ARIEL2.Conclusion: This pair of clinical trials seeks to determine prospectively the appropriate population, among all women with platinum-sensitive OC, who derive a meaningful benefit from PARPi treatment in the maintenance setting. Citation Format: Elizabeth Swisher, Iain McNeish, Robert L. Coleman, James D. Brenton, Scott H. Kaufmann, Andrew Allen, Mitch Raponi, Heidi Giordano, Lara Maloney, Jeffrey Isaacson, Jonathan A. Ledermann. Prospective molecular identification of ovarian cancer patients benefiting from PARP inhibitor (PARPi, rucaparib) maintenance therapy - reaching beyond germline BRCA mutations. [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 CT339. doi:10.1158/1538-7445.AM2014-CT339