Kaufmann, Scott H.

3598 Background: Drug resistance to topotecan can be the result of BCRP/ABCG2 expression. BCRP is a member of the ABC transporter family that pumps anticancer drugs out of the cell. Lapatinib is a potent and selective dual inhibitor of epidermal growth factor receptor (EGFR or ErbB1) and ErbB2 (Her2/Neu). 4-aminoquinazoline tyrosine kinase inhibitors have been shown to enhance the cytotoxicity of topotecan through inhibition of BCRP-mediated drug efflux in cancer cells. Methods: Thirty-seven patients with advanced stage cancers were enrolled at escalating dose levels of lapatinib and topotecan in cohorts IA, IB and IIB (MTD). Treatment schedule included lapatinib (750 - 1500 mg/d) daily for 21 (cohort IA) or 28 days (cohort IB) and topotecan (2.4 - 4.0 mg/ m2), days 1, 8 and 15; cycles were repeated every 28 days. Three patients were treated at each dose level, 18 on cohort IA, 9 on cohort IB and 10 at MTD (cohort IIB). Assessments of toxicity were performed with each cycle and clinical response was determined per RECIST criteria every other cycle. Results: The MTD for cohorts IA and IB was reached at a dose of 1250 mg of lapatinib and 3.2 mg/m2 of IV topotecan on days 1, 8 and 15. No DLT were seen during the dose escalation stage of cohorts IA and IB. Ten patients were enrolled at the MTD. There were no grade 4+ events. Thirteen grade 3+ events, considered to be related to treatment, were seen in 6 patients. The most common grade 3+ toxicities included dehydration (2) diarrhea (2), nausea (3), vomiting (2), neutropenia (1), thrombocytopenia (1), and fatigue (1). No abnormalities in left ventricular ejection fraction were noted. Stable disease was seen in 46% of the 37 patients. Conclusions: The combination of lapatinib and topotecan is a well-tolerated regimen. The MTD for the combination is lapatinib 1,250 mg orally once daily for 21 or 28 days and topotecan 3.2 mg/m2 on days 1, 8 and 15. Pharmacokinetic analysis for drug interaction will be available for presentation at the meeting. Supported in part by GSK and Mayo Clinic No significant financial relationships to disclose.

Abstract Purpose: To determine if the degree of estrogen suppression with aromatase inhibitors (AI: anastrozole, exemestane, letrozole) is associated with efficacy in early-stage breast cancer, and to examine for differences in the mechanism of action between the three AIs. Experimental Design: Matched case–control studies [247 matched sets from MA.27 (anastrozole vs. exemestane) and PreFace (letrozole) trials] were undertaken to assess whether estrone (E1) or estradiol (E2) concentrations after 6 months of adjuvant therapy were associated with risk of an early breast cancer event (EBCE). Preclinical laboratory studies included luciferase activity, cell proliferation, radio-labeled ligand estrogen receptor binding, surface plasmon resonance ligand receptor binding, and nuclear magnetic resonance assays. Results: Women with E1 ≥1.3 pg/mL and E2 ≥0.5 pg/mL after 6 months of AI treatment had a 2.2-fold increase in risk (P = 0.0005) of an EBCE, and in the anastrozole subgroup, the increase in risk of an EBCE was 3.0-fold (P = 0.001). Preclinical laboratory studies examined mechanisms of action in addition to aromatase inhibition and showed that only anastrozole could directly bind to estrogen receptor α (ERα), activate estrogen response element-dependent transcription, and stimulate growth of an aromatase-deficient CYP19A1−/− T47D breast cancer cell line. Conclusions: This matched case–control clinical study revealed that levels of estrone and estradiol above identified thresholds after 6 months of adjuvant anastrozole treatment were associated with increased risk of an EBCE. Preclinical laboratory studies revealed that anastrozole, but not exemestane or letrozole, is a ligand for ERα. These findings represent potential steps towards individualized anastrozole therapy.

Abstract Purpose: Clear cell ovarian carcinoma (CCOC) is an aggressive disease that often demonstrates resistance to standard chemotherapies. Approximately 25% of patients with CCOC show a strong APOBEC mutation signature. Here, we determine which APOBEC3 enzymes are expressed in CCOC, establish clinical correlates, and identify a new biomarker for detection and intervention. Experimental Designs: APOBEC3 expression was analyzed by IHC and qRT-PCR in a pilot set of CCOC specimens (n = 9 tumors). The IHC analysis of APOBEC3B was extended to a larger cohort to identify clinical correlates (n = 48). Dose-response experiments with platinum-based drugs in CCOC cell lines and carboplatin treatment of patient-derived xenografts (PDXs) were done to address mechanistic linkages. Results: One DNA deaminase, APOBEC3B, is overexpressed in a formidable subset of CCOC tumors and is low or absent in normal ovarian and fallopian tube epithelial tissues. High APOBEC3B expression associates with improved progression-free survival (P = 0.026) and moderately with overall survival (P = 0.057). Cell-based studies link APOBEC3B activity and subsequent uracil processing to sensitivity to cisplatin and carboplatin. PDX studies extend this mechanistic relationship to CCOC tissues. Conclusions: These studies demonstrate that APOBEC3B is overexpressed in a subset of CCOC and, contrary to initial expectations, associated with improved (not worse) clinical outcomes. A likely molecular explanation is that APOBEC3B-induced DNA damage sensitizes cells to additional genotoxic stress by cisplatin. Thus, APOBEC3B is a molecular determinant and a candidate predictive biomarker of the therapeutic response to platinum-based chemotherapy. These findings may have broader translational relevance, as APOBEC3B is overexpressed in many different cancer types.

Abstract Germline loss-of-function mutations in BRCA1 interacting protein C-terminal helicase 1 (BRIP1) are associated with ovarian carcinoma and may also contribute to breast cancer risk, particularly among patients who develop disease at an early age. Normal BRIP1 activity is required for DNA interstrand cross-link (ICL) repair and is thus central to the maintenance of genome stability. Although pathogenic mutations have been identified in BRIP1, genetic testing more often reveals missense variants, for which the impact on molecular function and subsequent roles in cancer risk are uncertain. Next-generation sequencing of germline DNA in 2,160 early-onset breast cancer and 1,199 patients with ovarian cancer revealed nearly 2% of patients carry a very rare missense variant (minor allele frequency < 0.0001) in BRIP1. This is 3-fold higher than the frequency of all rare BRIP1 missense alleles reported in more than 60,000 individuals of the general population (P < 0.0001, χ2 test). Using CRISPR-Cas9 gene editing technology and rescue assays, we functionally characterized 20 of these missense variants, focusing on the altered protein's ability to repair ICL damage. A total of 75% of the characterized variants rendered the protein hypomorph or null. In a clinical cohort of >117,000 patients with breast and ovarian cancer who underwent panel testing, the combined OR associated with BRIP1 hypomorph or null missense carriers compared with the general population was 2.30 (95% confidence interval, 1.60–3.30; P < 0.0001). These findings suggest that novel missense variants within the helicase domain of BRIP1 may confer risk for both breast and ovarian cancer and highlight the importance of functional testing for additional variants. Significance: Functional characterization of rare variants of uncertain significance in BRIP1 revealed that 75% demonstrate loss-of-function activity, suggesting rare missense alleles in BRIP1 confer risk for both breast and ovarian cancer.

Background: Outcomes of patients with AML have remained poor despite the availability of cytotoxic chemotherapy, hypomethylating agents (HMAs) and targeted therapies. HMAs, such as azacitidine, in combination with Bcl-2 inhibitors like venetoclax have demonstrated response rates of 67% in newly diagnosed AML and 21% in relapsed/refractory (RR) AML (DiNardo et al. Blood 2019 and Am J Hematol 2018). While the combination of azacitidine and venetoclax is efficacious in AML, preclinical studies indicate potential mechanisms of drug resistance including overexpression of MCL-1, an anti-apoptotic protein (Konopleva et al. Cancer Cell. 2006). Pevonedistat is a first in class inhibitor of Nedd8 activating enzyme that has demonstrated activity against AML (Swords RT et al. Blood. 2010). Pevonedistat induces NOXA, a pro-apoptotic protein leading to neutralization of MCL-1 inducing apoptosis (Wang et al. Biochem Biophys Res Commun. 2017). Preclinical studies evaluating the combination of pevonedistat and venetoclax against AML cell lines have demonstrated synergistic effect (Knoor KL et al. Cell Death Differ. 2015). Hence, we hypothesize that the addition of pevonedistat to the combination of azacitidine and venetoclax would enhance the therapeutic efficacy by overcoming resistance to apoptosis. Study design and methods: This is an investigator-initiated phase Ib study evaluating the safety of pevonedistat, azacitidine and venetoclax. Patients aged 18 years or above with morphologically documented AML (de novo, secondary or therapy-related), ECOG performance status 0-2 and adequate organ function are eligible for the study. Major exclusion criteria are patients with isolated extramedullary relapse, hematopoietic cell transplantation (HCT) within 100 days of enrollment, active acute GVHD, veno-occlusive disease, acute promyelocytic leukemia, liver cirrhosis and severe liver impairment. While the dose escalation phase is available only for patients with RR-AML, the dose expansion phase can also include newly diagnosed AML patients who are ineligible for intensive induction. The study is planned to be conducted at Medical College of Wisconsin, Mayo Clinic and University of Pennsylvania. The primary endpoint is to determine the recommended phase 2 dose (RP2D) and toxicity profile of pevonedistat, azacitidine and venetoclax. The secondary endpoints include determination of response rates, duration of response, survival and pharmacokinetics. Exploratory endpoints include correlation of response rates with AML genomic profile, correlation of pretreatment levels of BCL2, BCLXL, MCL1, BAX or BAK with response, determination of changes in NOXA (PMAIP1) mRNA and protein expression pre-and post-pevonedistat treatment, evaluation of BH3 mimetic profiling on bone marrow samples by flow cytometry and assessing the sensitivity of leukemia and leukemic stem/progenitor cells to pevonedistat ex vivo. The study will follow 3+3 design with dose escalation (Arms A and B), de-escalation in case of dose limiting toxicity (DLT) (arms Z and Y) and dose expansion phase (figure 1). Patients will be entered sequentially to each dose level, starting with dose level 0. The DLT observation period for dose-escalation will be 1 cycle. The maximal tolerated dose (MTD) will be defined as the highest dose level at which none of the first 3 treated subjects, or no more than 1 of the first 6 treated subjects experiences a DLT. A minimum of 9 and a maximum of 24 patients will be needed for the dose escalation phase and 6 patients for the dose expansion phase. Response rate, duration of response and exploratory endpoints will be analyzed using descriptive statistics. Kaplan-Meier method will be used to determine survival. Disclosures Guru Murthy: Cardinal Health Inc.: Honoraria. Michaelis:Incyte: Consultancy, Research Funding; Pfizer: Equity Ownership, Research Funding; Novartis: Consultancy; Macrogeneics: Research Funding; Millenium: Research Funding; BMS: Research Funding; Celgene: Consultancy, Research Funding; JAZZ: Other: Data Safety Monitoring Board, uncompensated, Research Funding; TG Therapeutics: Consultancy, Research Funding; Janssen: Research Funding; ASTEX: Research Funding; Bioline: Research Funding. Abedin:Jazz Pharmaceuticals: Honoraria; Agios: Honoraria; Helsinn Healthcare: Research Funding; Pfizer Inc: Research Funding; Actinium Pharmaceuticals: Research Funding. Runaas:Agios: Honoraria; Blueprint Medicine: Honoraria. Atallah:Jazz: Consultancy; Novartis: Consultancy; Takeda: Consultancy, Research Funding; Pfizer: Consultancy; Jazz: Consultancy; Helsinn: Consultancy; Helsinn: Consultancy.

Introduction: Chronic myelomonocytic leukemia (CMML), an aggressive myeloid malignancy, can be categorized into two subtypes, proliferative CMML (pCMML) and dysplastic (dCMML), based on a white blood cell (WBC) count of ≥ 13 x 109/L for the former (Arber et al. Blood 2016). While this WBC cut off is somewhat arbitrary, patients with pCMML have unique phenotypic features and a shorter survival. We carried out this study to assess the genomic, transcriptomic and epigenetic landscapes of these two CMML subtypes. Methods: Peripheral blood (PB) and bone marrow (BM) mononuclear cells (MNC) were obtained from WHO-defined CMML patients. Next generation sequencing (NGS) using a 36-gene panel was performed on 350 patients with Illumina HiSeq4000 platform with median read depth of 400X. RNA sequencing (RNA-seq) was performed on 25 patients by bulk whole transcriptome sequencing using Illumina TruSeq. DNA immunoprecipitation sequencing (DIP-seq) was performed on 18 patients using 5-methylcystocine (5mC), 5-hydroxymethylcytosine (5hmC) and bridging monoclonal antibodies with subsequent paired-end sequencing using HiSeq4000. Chromatin immunoprecipitation sequencing (ChIP-seq) was performed on 30 patients with Illumina HiSeq2500 to a depth of 25 million for histone 3 lysine 4 monomethylation (H3K4me1) and histone 3 lysine 4 trimethylation (H3K4me3) and 50 million reads for histone 3 lysine 27 trimethylation (H3K27me3) and Input per sample. Results: Five hundred and seventy-three patients with WHO defined CMML were included; median age 71 years (range 18-95 years), 67% males. 282 patients had pCMML (49%), while 291 (51%) had dCMML. As pre-defined, patients with pCMML were more likely to have higher absolute monocyte counts (p<0.0001), circulating immature myeloid cells (p<0.0001), PB blasts (p<0.0001), and higher lactate dehydrogenase levels (p=0.03). At last follow up 234 (41%) deaths and 70 (20%) leukemic transformations were documented. The median OS for pCMML vs dCMML in this cohort was 19 months vs 30 months (p<0.0001, Figure 1A) and validated in an independent Austrian cohort (p=0.02). Genomic profiling: NGS performed on 350 patients (BM MNC) revealed a higher frequency of NRAS (35 vs 17%, p=0.004), cumulative RAS pathway (NRAS, KRAS, CBL and PTPN11) (73 vs 47%, p=0.001), ASXL1 (p=0.003) and JAK2V617F (p=0.04) mutations in pCMML relative to dCMML (Figure 1B); while dCMML had a higher frequency of SF3B1 mutations (p=0.02). There were no differences in distribution of TET2 and SRSF2Transcriptomic analysis: RNA-seq was performed on PB MNC from RAS pathway mutant pCMML patients (n=12) and RAS pathway wildtype dCMML patients (n=13). Unsupervised clustering analysis resulted in appropriate segregation revealing distinct expression profiles between disease subtypes (Figure 1C). Compared to dCMML, 3729 genes were significantly differentially upregulated and 2658 genes were differentially downregulated in pCMML. Among genes most highly upregulated were mitotic checkpoint kinases including AURBK, PLK1, PLK2, PLK4 andEpigenetic profiling: ChIP-seq of PB and BM MNC from pCMML (n=18) and dCMML (n=12) patients and healthy, age-matched controls (n=10) revealed a global increase in H3K4me1, without significant differences in H3K4me3 or H3K27me3 occupancies (regardless of stratification by ASXL1 mutational status; 40% ASXL1mt in pCMML, 30% dCMML) in pCMML vs dCMML (Figure 1D). H3K4me1 occupancy was also increased in a sequence-specific manner at the transcription start sites of the aforementioned mitotic kinases (PLK1 and WEE1). DIP-seq was performed on PB MNC to assess global differences in 5-mC and 5-hmC levels, between pCMML (n=9) and dCMML (n=9), with no differences seen between the two subtypes (regardless of TET2 mutational status, 40% TET2mt in each subtype) (Figure 1E). Conclusions: Despite the somewhat arbitrary WBC distinction between pCMML and dCMML, clear phenotypic, genetic, transcriptomic, epigenetic and survival differences exist between the two subtypes, providing strong biological rationale for this distinction. pCMML patients have a higher frequency of oncogenic RAS pathway mutations, a unique transcriptomic profile enriched in mitotic check point kinases and a unique chromatin configuration with global and sequence specific enrichment in H3K4me1, with no significant global differences in 5mC, 5hmC, or H3K4me3 and H3K27me3 occupancy. Figure 1 Disclosures Geissler: AOP: Honoraria; Pfizer: Honoraria; AstraZeneca: Honoraria; Novartis: Honoraria; Celgene: Honoraria; Roche: Honoraria; Abbvie: Honoraria; Ratiopharm: Honoraria; Amgen: Honoraria. Al-Kali:Astex Pharmaceuticals, Inc.: Research Funding. Patnaik:Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees.

Abstract BRCA1 plays a key role in homologous recombination (HR) DNA repair. Accordingly, changes that downregulate BRCA1, including BRCA1 mutations and reduced BRCA1 transcription, due to promoter hypermethylation or loss of the BRCA1 transcriptional regulator CDK12, disrupt HR in multiple cancers. In addition, BRCA1 has also been implicated in the regulation of metabolism. Here, we show that reducing BRCA1 expression, either by CDK12 or BRCA1 depletion, led to metabolic reprogramming of ovarian cancer cells, causing decreased mitochondrial respiration and reduced ATP levels. BRCA1 depletion drove this reprogramming by upregulating nicotinamide N-methyltransferase (NNMT). Notably, the metabolic alterations caused by BRCA1 depletion and NNMT upregulation sensitized ovarian cancer cells to agents that inhibit mitochondrial metabolism (VLX600 and tigecycline) and to agents that inhibit glucose import (WZB117). These observations suggest that inhibition of energy metabolism may be a potential strategy to selectively target BRCA1-deficient high-grade serous ovarian cancer, which is characterized by frequent BRCA1 loss and NNMT overexpression. Significance: Loss of BRCA1 reprograms metabolism, creating a therapeutically targetable vulnerability in ovarian cancer.

Abstract Introduction: Topoisomerase I is a nuclear enzyme that relaxes torsional strain in DNA by nicking one DNA strand, allowing rotation, then resealing the DNA backbone. This is an important enzyme in replication and transcription, and therefore has been targeted by anti-cancer agents such as camptothecins. These agents intercalate into DNA at the topoisomerase I active site and inhibit the religation step catalyzed by the enzyme, causing increased topoisomerase I-DNA complexes that impede advancing replication forks or transcription complexes, leading to DNA damage and cell death. Platinating agents also stabilize topoisomerase I-DNA covalent complexes. The topotecan/carboplatin combination has recently shown promise in combination with the PARP inhibitor veliparib in aggressive and transformed myeloproliferative neoplasms (MPNs) and chronic myelomonocytic leukemia (K Pratz et al., Clin Cancer Res. 23:899, 2017). Given that both classes of agents have a narrow therapeutic window, it is important to try to predict the sensitivity/resistance of these agents in malignancies such as acute myeloid leukemia (AML) and transformed MPNs. Some of mechanisms of drug resistance that have been described are decreased numbers of drug-stabilized topoisomerase I-DNA complexes due to drug efflux, diminished topoisomerase I expression or topoisomerase I mutation. Thus, being able to detect and quantify these DNA-protein complexes may provide insight into drug sensitivity. Methods: Using a unique monoclonal antibody that detects topoisomerase I-DNA covalent complexes (A. Patel et al., Nucleic Acids Res. 44:2816, 2016), immunofluorescence techniques were used to quantify these topoisomerase I-DNA complexes in AML cell lines (SET2, HEL, K562, U937, ML2, MV-4-11, THP1, HL60, ML1, and Molm13) and clinical AML samples. To verify the fidelity of the anti-topoisomerase I-DNA covalent complex antibody (TopIcc), siRNA knockdown methods were utilized with confirmatory immunofluorescence and Western blotting. Slot blots were additionally used to assess the presence of these complexes in AML cell lines. Western blots were performed to assess topoisomerase I levels in AML cell lines. Topotecan drug sensitivity analysis was performed by using an MTS assay. Results: With treatment of the topoisomerase I inhibitor, topotecan, there was an increase in the percentage of leukemia cells positive for the topoisomerase I-DNA covalent complexes. The degree of increase in complexes varied across cell lines and patient samples. To ensure we were detecting TopIcc by immunofluorescence, we performed a topoisomerase-I siRNA knockdown to confirm the specificity of the antibody for TopIcc's. Slot blots on multiple AML cell lines confirmed the presence of topoisomerase-DNA covalent complexes. The Western blot of all the AML cell lines, ordered from low to high TopIcc revealed increasing amounts of topoisomerase I. The MTS assay revealed lower topotecan IC50 values in three of the cell lines with higher complex formation, including Molm13, ML1 and ML2 (IC50 6-8 nM compared to 13-46 nM in the other cell lines). Conclusions: Based on recent clinical results, there has been renewed interested in topoisomerase I inhibitors, especially for aggressive and transformed MPNs. Topoisomerase I-DNA complex repair/downregulation has been a hallmark of drug resistance to topoisomerase I inhibitors. Therefore, detecting and quantifying these complexes in treated samples has been hypothesized to provide insight into the drug sensitivity, which is particularly important in camptothecins and platinating agents given their narrow therapeutic windows. Immunodetection techniques using an anti-TopIcc antibody may help predict sensitivity of AML to topoisomerase I inhibitors and will be used as a correlative biomarker study in an upcoming phase II randomized study of topotecan/carboplatin with or without veliparib or placebo in advanced myeloproliferative disorders and chronic myelomonocytic leukemia (ClinicalTrials.gov NCT03289910). Disclosures Pratz: AbbVie: Consultancy, Research Funding; Agios: Research Funding; Astellas: Consultancy, Research Funding; Boston Scientific: Consultancy; Millenium/Takeda: Research Funding. Kaufmann:Mayo Clinic: Patents & Royalties: Co-inventor on a patent held by Mayo Clinic for the use of the anti-TOP1cc antibody as a diagnostic reagent.

Abstract Treatment of HL-60 human leukemia cells with etoposide induces apoptotic cell death and activation of at least 18 electrophoretically distinct cysteine-dependent aspartate-directed protease (caspase) isoforms, several of which differ only in their isoelectric points. The purpose of the present study was to determine whether activated caspases are phosphorylated. Phosphatase treatment of cytosolic extracts containing active caspases followed by affinity labeling with N-(N-benzyloxycarbonylglutamyl-N-biotinyllysyl)aspartic acid [(2,6-dimethylbenzoyl)oxy] methyl ketone (Z-EK(bio)D-aomk) showed a mobility shift in several of the labeled species, suggesting that phosphorylated forms of these enzymes are present in the extracts. Metabolic labeling with 32P followed by etoposide treatment and subsequent affinity purification of affinity-labeled caspases confirmed that at least three caspase species were phosphorylated. To detect effects of the phosphorylation on enzymatic activity, caspase-mediated cleavage of aspartylglutamylvalinylaspartyl-7-amino-4-trifluoromethylcoumarin (DEVD-AFC) and poly(ADP-ribose) polymerase (PARP) by phosphorylated and dephosphorylated extracts was measured. No significant changes in Km or vmax were detected using DEVD-AFC. In contrast, a slight, but significant enhancement of PARP cleavage was observed in dephosphorylated extracts, suggesting that phosphorylation of active caspases could have an inhibitory effect on enzyme activity. These observations, which provide the first evidence that caspases are phosphoproteins, suggest that caspases may be targets for some of the growing list of protein kinases that are involved in apoptotic events. © 1998 by The American Society of Hematology.