Deborah Wilsker

Phase I Study of Single-Agent AZD1775 (MK-1775), a Wee1 Kinase Inhibitor, in Patients With Refractory Solid Tumors

PURPOSE Wee1 tyrosine kinase phosphorylates and inactivates cyclin-dependent kinase (Cdk) 1/2 in response to DNA damage. AZD1775 is a first-in-class inhibitor of Wee1 kinase with single-agent antitumor activity in preclinical models. We conducted a phase I study of single-agent AZD1775 in adult patients with refractory solid tumors to determine its maximum-tolerated dose (MTD), pharmacokinetics, and modulation of phosphorylated Tyr15-Cdk (pY15-Cdk) and phosphorylated histone H2AX (γH2AX) levels in paired tumor biopsies. PATIENTS AND METHODS AZD1775 was administered orally twice per day over 2.5 days per week for up to 2 weeks per 21-day cycle (3 + 3 design). At the MTD, paired tumor biopsies were obtained at baseline and after the fifth dose to determine pY15-Cdk and γH2AX levels. Six patients with BRCA-mutant solid tumors were also enrolled at the MTD. RESULTS Twenty-five patients were enrolled. The MTD was established as 225 mg twice per day orally over 2.5 days per week for 2 weeks per 21-day cycle. Confirmed partial responses were observed in two patients carrying BRCA mutations: one with head and neck cancer and one with ovarian cancer. Common toxicities were myelosuppression and diarrhea. Dose-limiting toxicities were supraventricular tachyarrhythmia and myelosuppression. Accumulation of drug (t1/2 approximately 11 hours) was observed. Reduction in pY15-Cdk levels (two of five paired biopsies) and increases in γH2AX levels (three of five paired biopsies) were demonstrated. CONCLUSION This is the first report of AZD1775 single-agent activity in patients carrying BRCA mutations. Proof-of-mechanism was demonstrated by target modulation and DNA damage response in paired tumor biopsies.

The National Cancer Institute ALMANAC: A Comprehensive Screening Resource for the Detection of Anticancer Drug Pairs with Enhanced Therapeutic Activity

To date, over 100 small-molecule oncology drugs have been approved by the FDA. Because of the inherent heterogeneity of tumors, these small molecules are often administered in combination to prevent emergence of resistant cell subpopulations. Therefore, new combination strategies to overcome drug resistance in patients with advanced cancer are needed. In this study, we performed a systematic evaluation of the therapeutic activity of over 5,000 pairs of FDA-approved cancer drugs against a panel of 60 well-characterized human tumor cell lines (NCI-60) to uncover combinations with greater than additive growth-inhibitory activity. Screening results were compiled into a database, termed the NCI-ALMANAC (A Large Matrix of Anti-Neoplastic Agent Combinations), publicly available at https://dtp.cancer.gov/ncialmanac Subsequent in vivo experiments in mouse xenograft models of human cancer confirmed combinations with greater than single-agent efficacy. Concomitant detection of mechanistic biomarkers for these combinations in vivo supported the initiation of two phase I clinical trials at the NCI to evaluate clofarabine with bortezomib and nilotinib with paclitaxel in patients with advanced cancer. Consequently, the hypothesis-generating NCI-ALMANAC web-based resource has demonstrated value in identifying promising combinations of approved drugs with potent anticancer activity for further mechanistic study and translation to clinical trials. Cancer Res; 77(13); 3564-76. ©2017 AACR.

Translating pharmacodynamic biomarkers from bench to bedside: analytical validation and fit-for-purpose studies to qualify multiplex immunofluorescent assays for use on clinical core biopsy specimens

Multiplex pharmacodynamic (PD) assays have the potential to increase sensitivity of biomarker-based reporting for new targeted agents, as well as revealing significantly more information about target and pathway activation than single-biomarker PD assays. Stringent methodology is required to ensure reliable and reproducible results. Common to all PD assays is the importance of reagent validation, assay and instrument calibration, and the determination of suitable response calibrators; however, multiplex assays, particularly those performed on paraffin specimens from tissue blocks, bring format-specific challenges adding a layer of complexity to assay development. We discuss existing multiplex approaches and the development of a multiplex immunofluorescence assay measuring DNA damage and DNA repair enzymes in response to anti-cancer therapeutics and describe how our novel method addresses known issues.

Development of a quantitative pharmacodynamic assay for apoptosis in fixed tumor tissue and its application in distinguishing cytotoxic drug−induced DNA double strand breaks from DNA double strand breaks associated with apoptosis

DNA double strand breaks (DSBs) induced by cancer therapeutic agents can lead to DNA damage repair or persistent DNA damage, which can induce apoptotic cell death; however, apoptosis also induces DSBs independent of genotoxic insult. γH2AX is an established biomarker for DSBs but cannot distinguish between these mechanisms. Activated cleaved caspase-3 (CC3) promotes apoptosis by enhancing nuclear condensation, DNA fragmentation, and plasma membrane blebbing. Here, we describe an immunofluorescence assay that distinguishes between apoptosis and drug-induced DSBs by measuring coexpression of γH2AX and membrane blebbing−associated CC3 to indicate apoptosis, and γH2AX in the absence of CC3 blebbing to indicate drug-induced DNA damage. These markers were examined in xenograft models following treatment with topotecan, cisplatin, or birinapant. A topotecan regimen conferring tumor regression induced tumor cell DSBs resulting from both apoptosis and direct DNA damage. In contrast, a cisplatin regimen yielding tumor growth delay, but not regression, resulted in tumor cell DSBs due solely to direct DNA damage. MDA-MB-231 xenografts exposed to birinapant, which promotes apoptosis but does not directly induce DSBs, exhibited dose-dependent increases in colocalized γH2AX/CC3 blebbing in tumor cells. Clinical feasibility was established using formalin-fixed, paraffin-embedded biopsies from a canine cancer clinical trial; γH2AX/CC3 colocalization analysis revealed apoptosis induction by two novel indenoisoquinoline topoisomerase I inhibitors, which was consistent with pathologist-assessed apoptosis and reduction of tumor volume. This assay is ready for use in clinical trials to elucidate the mechanism of action of investigational agents and combination regimens intended to inflict DNA damage, apoptotic cell death, or both.

Abstract 3072: A clinical pharmacodynamic biomarker assay that distinguishes potentially repairable, cytotoxic drug-induced DNA double strand breaks (DSBs) from DSBs associated with apoptotic cell death

The effectiveness of certain classes of cytotoxic cancer therapeutics likely depends on whether drug-induced DNA damage is successfully repaired or not, with the latter situation leading to mutations and strand breaks. However, double strand breaks (DSBs) also occur independently of genotoxic insults during apoptotic cell death caused by many drug classes, as well as natural biological processes. We have developed an immunofluorescent confocal microscopy assay that uses a biomarker profile suitable for individual cell analysis designed to distinguish between DSBs caused by apoptosis and those caused by direct DNA damage from cytotoxic drug action. γ-H2AX is an established biomarker for DSBs and activated cleaved caspase 3 is an executioner caspase important for apoptosis, which leads to nuclear condensation, DNA fragmentation, plasma membrane blebbing, and subsequent cell death. Our assay defines the DSBs of apoptosis by co-localized γ-H2AX and cleaved caspase 3 in individual cells, while defining the DSBs from early drug effects of DNA damaging chemotherapeutics by γ-H2AX induction in the absence of cleaved caspase 3. Building on our published findings that topotecan strongly induces γ-H2AX and DSBs within 1-4 hours in vitro and in vivo, we observed exposure-dependent increases in γ-H2AX /cleaved caspase 3 double positive cells at later time points, both in an HT29 in vitro spheroid model and an MDA-MB-231 xenograft model. Fit-for-purpose studies in the MDA-MB-231 xenograft model treated with birinipant, a SMAC mimetic and IAP deregulator that does not directly produce lethal DSBs, demonstrated a dose-dependent increase in cellular co-localization of γ-H2AX/cleaved caspase 3 consistent with birinipant induced apoptosis and the established mechanism of action of this compound. Clinical feasibility was established in a canine clinical trial using formalin-fixed paraffin-embedded (FFPE) 18-gauge needle biopsies: two novel indenoisoquinolines, indotecan (LMP400) and indimitecan (LMP776), increased tumor cell co-localization of γ-H2AX/cleaved caspase 3 in tumor samples obtained on day 5 of qdx5 treatment. This PD biomarker assay of early and late DSB response to drug exposure could have important applications for elucidation of mechanisms of action of anticancer drugs and the development of investigational agents. Funded by NCI Contract No. HHSN261200800001E. Citation Format: Angie B. Dull, Deborah Wilsker, Robert J. Kinders, Ralph E. Parchment, David Evans, Beverly A. Teicher, James H. Doroshow. A clinical pharmacodynamic biomarker assay that distinguishes potentially repairable, cytotoxic drug-induced DNA double strand breaks (DSBs) from DSBs associated with apoptotic cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3072. doi:10.1158/1538-7445.AM2017-3072

Abstract 4678: Pilot trial of talazoparib (BMN 673), an oral PARP inhibitor, in patients with advanced solid tumors carrying deleterious BRCA mutations

Inhibition of poly (ADP-ribose) polymerase (PARP) sensitizes tumor cells to DNA damage that would normally be repaired through the base excision repair pathway. PARP inhibitors are active clinically against BRCA-deficient ovarian cancers. The PARP inhibitor talazoparib produces cytotoxicity in human cancer cell lines and animal models of tumors that harbor mutations that compromise DNA repair pathways. In this study, single agent talazoparib (1000 µg/day) was administered to patients with deleterious BRCA1 or BRCA2 mutations and advanced solid tumors in 28 day cycles. The primary objective of the trial was to examine pharmacodynamic (PD) effects of talazoparib; the secondary objective was to determine response rate in patients whose tumors carry BRCA mutations. Mandatory paired tumor biopsies were obtained pre-treatment and 3-6 hrs post-treatment on cycle 1 day 8. Optional biopsies were collected at the time of progression. One core from each time point was analyzed for PARP inhibition by a validated ELISA assay while the other core was used for IFA analysis of γH2AX. A total of 9 patients (pts) were enrolled and treated before this trial was closed due to lack of drug availability: [prostate (3), ovarian (2), breast (2), uterine sarcoma (1), pancreatic (1)]. Median age was 63 (range: 33-73 yrs); male-to-female ratio was 4:5; and the median number of prior treatments was 6 (range: 1-12). All 9 pts were evaluable for PD endpoints. One pt progressed during the first cycle of treatment; 8 pts were evaluable for clinical response. Mean time on study for evaluable pts was 8 cycles (range: 2-18); 5 of 8 (62%) pts experienced a documented partial responses [ovarian (2), prostate (2), breast (1)] lasting between 4 and 12 cycles (median: 6 cycles). Two pts had stable disease for 4 to 6 cycles, and one progressed after 2 cycles. The agent was well tolerated; the most frequent adverse events were hematologic including grade (gr) 4 anemia (1) and thrombocytopenia (1), and gr 3 anemia (2), neutropenia (1), lymphopenia (1). Decreases in PAR levels (>75%) were observed in all cycle 1 day 8 biopsy pairs, documenting a primary PD effect. Increased γH2AX expression was observed for 4/6 pts in post-dose biopsies; pre-treatment γH2AX levels, measured as %Nuclear Associated Protein (NAP), increased from a mean ± SD of 1.33 ± 1.08 to a post-treatment %NAP mean of 5.60 ± 0.78; (p=0.018), supporting a role for drug-enhanced DNA double strand breaks in the mechanism of action of talazoparib for BRCA mutant tumors. In summary, talazoparib demonstrated significant clinical activity as a single agent in patients with BRCA-deficient tumors and produced substantial reductions in tumor PAR levels in matched pre and post-treatment tumor biopsies. Citation Format: Robert S. Meehan, Alice P. Chen, Geraldine O’Sullivan Coyne, Shivaani Kummar, Jiuping Ji, Rasa Vilimas, Lamin Juwara, Robert J. Kinders, Katherine Ferry-Galow, Deborah Wilsker, Yiping Zhang, Angie B. Dull, Tony Navas, Lihua Wang, Ralph E. Parchment, James H. Doroshow. Pilot trial of talazoparib (BMN 673), an oral PARP inhibitor, in patients with advanced solid tumors carrying deleterious BRCA mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4678. doi:10.1158/1538-7445.AM2017-4678

The root causes of pharmacodynamic assay failure

Robust pharmacodynamic assay results are valuable for informing go/no-go decisions about continued development of new anti-cancer agents and for identifying combinations of targeted agents, but often pharmacodynamic results are too incomplete or variable to fulfill this role. Our experience suggests that variable reagent and specimen quality are two major contributors to this problem. Minimizing all potential sources of variability in procedures for specimen collection, processing, and assay measurements is essential for meaningful comparison of pharmacodynamic biomarkers across sample time points. This is especially true in the evaluation of pre- and post-dose tumor biopsies, which suffer from high levels of tumor insufficiency due to variations in biopsy collection techniques and significant specimen heterogeneity within and across patients. Developing methods to assess heterogeneous biopsies is necessary in order to evaluate a majority of tumor biopsies collected for pharmacodynamic biomarker studies. Improved collection devices and standardization of methods are being sought in order to improve the tumor content and quality of tumor biopsies. In terms of reagent variability, we have found that stringent initial reagent qualification and quality control of R&D-grade reagents is critical to minimize lot-to-lot variability and prevent assay failures, especially for clinical pharmacodynamic questions, which often demand assay performance that meets or exceeds clinical diagnostic assay standards. Rigorous reagent specifications and use of appropriate assay quality control methodologies help to ensure consistency between assay runs, laboratories and trials to provide much needed pharmacodynamic insights into the activity of investigational agents.

Abstract B24: Ataxia-telangiectasia and Rad3-related (ATR) phosphorylation as a pharmacodynamic biomarker of ATR activation in solid tumor tissue models

Inhibitors of checkpoint kinases, such as ATR, Chk1 or Wee1, in combination with cytotoxic agents could enhance therapeutic efficacy compared to monotherapy, and these combination approaches are currently being extensively explored. The presence of replicative stress or deregulated S-phase in cancer has been recognized as a rationale for the use of ATR and Chk1 inhibitors with chemotherapy and efforts are underway to define genetic determinants that sensitize cancer cells to ATR inhibition. Pharmacodynamic (PD) biomarkers of drug activity are valuable tools in clinical trials using targeted agents to determine whether each investigational agent is acting upon its intended target, the expected intracellular signaling pathways are modulated, and the dosage regimen and sequencing are optimal. In addition, investigations of the potential effects of cell cycle distribution within the tumor (in animal models) on drug efficacy may point to alternative dosing regimens. The first clinical trials evaluating inhibitors of the ataxia telangiectasia and Rad3-related kinase (ATR) in combination with cytotoxic agents are enrolling patients. The current method for quantifying ATR inhibition used in preclinical in vitro and in vivo models is via indirect measurement of downstream effector proteins, such as phosphorylated Chk1, rather than measurement of direct target engagement. We propose that an immunofluorescence microscopy assay of ATR autophosphorylation status as a biomarker of DNA damage activation combined with measurement of the downstream effector proteins will reveal more specific information about target engagement and intended PD effect than would be obtained from a single-biomarker assay. Here we propose that the measurement of ATR pT1989, Chk1 pS345 and γH2AX by validated quantitative immunofluorescence assays will be useful in PD evaluation of tumor responses in clinical trials as a biomarker for ATR pathway activation or inhibition. In addition, evaluation of the variability of expression of activated ATR in tumor models of different histologies may provide valuable insight into tumor types likely to be sensitive to ATR inhibitors. Citation Format: Deborah Wilsker, Allison M. Marrero, Angie Dull, Thomas D. Pfister, Scott M. Lawrence, John Carter, Michelle Gottholm-Ahalt, Melinda Hollingshead, James Doroshow, Ralph E. Parchment, Robert J. Kinders. Ataxia-telangiectasia and Rad3-related (ATR) phosphorylation as a pharmacodynamic biomarker of ATR activation in solid tumor tissue models. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr B24.

Abstract 4689: Pre-clinical investigation of the wee1 inhibitor MK-1775 using pharmacodynamic and mechanistic markers in diverse cancer models in vivo

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Wee1 kinase is a critical regulator of the G2/M checkpoint by initiating inhibitory phosphorylation of the conserved tyrosine-15 residue of cdk1/cdc2. Wee1 also phosphorylates tyrosine 15 of cdk2 and plays a crucial role in maintaining genome integrity during S-phase. MK1775 (NSC 754352) is a small molecule selective inhibitor of Wee1 kinase that is currently under clinical evaluation. We have developed a quantitative immunofluorescence assay to measure inhibition of Wee1 by detection of pY15 of cyclin-dependent kinases. A full analysis of the pharmacodynamic (PD) effects of Wee1 inhibition by MK1775, as well as the downstream mechanistic consequences of Wee1 inhibition on DNA repair, apoptosis, and premature mitotic entry markers was performed using two xenograft models: A673 Ewing sarcoma and U87-MG glioblastoma. Four DNA repair markers (pNbs1, γH2AX, Rad51, and pATR) were examined as well as the mitotic marker pHistone H3. Advanced quantitative image analysis was performed using Definiens software to measure changes in PD markers by two approaches: total nuclear area measurements and foci per nucleus. Definiens software enables enhanced analysis of the markers by enumeration of the nuclei in the imaged xenograft tissues over an entire data set with high-content capacity. Our data demonstrate greater than 80% inhibition of pY15-cdk in vivo at the clinically relevant dose of 60 mg/kg (180 mg/m2) MK1775 in two xenograft models. In addition, γH2AX induction was observed after multiple doses of MK1775 as a single agent. A PD biomarker time course was determined for MK1775 and gemcitabine (NSC 750927) as single agents to develop a drug administration schedule for combination studies in a Ewing sarcoma model. The time point at which the DNA damage response peaked following administration of the maximum tolerated dose of gemcitabine in the mouse was determined. This study elucidates a broad profile of PD marker response, as well as the corresponding levels of MK1775 in the xenografts. Funded by NCI Contract No. HHSN261200800001E. Citation Format: Deborah F. Wilsker, Allison M. Marrero, Melinda Hollingshead, Scott M. Lawrence, Alice Chen, Shivaani Kummar, Joseph M. Covey, Ralph E. Parchment, Robert J. Kinders, Joseph E. Tomaszewski, James H. Doroshow. Pre-clinical investigation of the wee1 inhibitor MK-1775 using pharmacodynamic and mechanistic markers in diverse cancer models in vivo . [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 4689. doi:10.1158/1538-7445.AM2014-4689