Benjamin J. Drapkin

Disruption of Mechanisms That Prevent Rereplication Triggers a DNA Damage Response

ABSTRACT Eukaryotes replicate DNA once and only once per cell cycle due to multiple, partially overlapping mechanisms efficiently preventing reinitiation. The consequences of reinitiation are unknown. Here we show that the induction of rereplication by mutations in components of the prereplicative complex (origin recognition complex [ORC], Cdc6, and minichromosome maintenance proteins) causes a cell cycle arrest with activated Rad53, a large-budded morphology, and an undivided nucleus. Combining a mutation disrupting the Clb5-Orc6 interaction (ORC6-rxl) and a mutation stabilizing Cdc6 (CDC6ΔNT) causes a cell cycle delay with a similar phenotype, although this background is only partially compromised for rereplication control and does not exhibit overreplication detectable by fluorescence-activated cell sorting. We conducted a systematic screen that identified genetic requirements for the viability of these cells. ORC6-rxl CDC6ΔNT cells depend heavily on genes required for the DNA damage response and for double-strand-break repair by homologous recombination. Our results implicate an Mre11-Mec1-dependent pathway in limiting the extent of rereplication.

Analysis of the mitotic exit control system using locked levels of stable mitotic cyclin

Cyclin‐dependent kinase (Cdk) both promotes mitotic entry (spindle assembly and anaphase) and inhibits mitotic exit (spindle disassembly and cytokinesis), leading to an elegant quantitative hypothesis that a single cyclin oscillation can function as a ratchet to order these events. This ratchet is at the core of a published ODE model for the yeast cell cycle. However, the ratchet model requires appropriate cyclin dose–response thresholds. Here, we test the inhibition of mitotic exit in budding yeast using graded levels of stable mitotic cyclin (Clb2). In opposition to the ratchet model, stable levels of Clb2 introduced dose‐dependent delays, rather than hard thresholds, that varied by mitotic exit event. The ensuing cell cycle was highly abnormal, suggesting a novel reason for cyclin degradation. Cdc14 phosphatase antagonizes Clb2–Cdk, and Cdc14 is released from inhibitory nucleolar sequestration independently of stable Clb2. Thus, Cdc14/Clb2 balance may be the appropriate variable for mitotic regulation. Although our results are inconsistent with the aforementioned ODE model, revision of the model to allow Cdc14/Clb2 balance to control mitotic exit corrects these discrepancies, providing theoretical support for our conclusions.

Trends in all-cause mortality among patients with chronic myeloid leukemia: a Surveillance, Epidemiology, and End Results database analysis.

Outcomes for patients with chronic myeloid leukemia (CML) have improved after the advent of tyrosine kinase inhibitors (TKIs), which target the BCR/ABL fusion gene product. Nonetheless, differences in survival persist between age groups. The authors performed a retrospective cohort study using the Surveillance, Epidemiology, and End Results (SEER) database to assess 5‐year overall survival (OS) in various patient age groups.

Association between baseline body mass index and overall survival among patients over age 60 with acute myeloid leukemia

Acute myeloid leukemia (AML) is more common and more lethal among patients over the age of 60. Increased body mass index (BMI) has been associated with a higher incidence of various malignancies, including AML. We sought to determine whether patient BMI at the time of AML diagnosis is related to overall survival (OS) among elderly patients. We identified 97 patients with AML diagnosed after the age of 60 and treated with cytarabine‐based induction chemotherapy. The median age was 68 years (range 60–87); 52% of patients were male, and our study population was predominantly white (89% of patients). The median OS for all patients was 316 days (95% CI 246–459). The hazard ratio for mortality was increased among patients with a BMI < 25 compared to BMI ≥ 30 (HR 2.14, P = 0.009, 95% CI 1.21–3.77), as well as with older age (HR 1.76, P = 0.015, 95% CI 1.12–2.79) and with secondary versus de novo disease (HR 1.95, P = 0.006, 95% CI 1.21–3.14). After multivariable analysis, we did not find a significant association between OS and other potential confounders such as coronary artery disease or diabetes among these patients. We conclude that increased BMI was independently associated with improved OS among older AML patients at our institution. Am. J. Hematol. 88:642–646, 2013.

Cyclin and Cyclin-Dependent Kinase Substrate Requirements for Preventing Rereplication Reveal the Need for Concomitant Activation and Inhibition

DNA replication initiation in S. cerevisiae is promoted by B-type cyclin-dependent kinase (Cdk) activity. In addition, once-per-cell-cycle replication is enforced by cyclin-Cdk-dependent phosphorylation of the prereplicative complex (pre-RC) components Mcm2-7, Cdc6, and Orc1-6. Several of these controls must be simultaneously blocked by mutation to obtain rereplication. We looked for but did not obtain strong evidence for cyclin specificity in the use of different mechanisms to control rereplication: both the S-phase cyclin Clb5 and the mitotic cyclins Clb1–4 were inferred to be capable of imposing ORC-based and MCM-based controls. We found evidence that the S-phase cyclin Clb6 could promote initiation of replication without blocking reinitiation, and this activity was highly toxic when the ability of other cyclins to block reinitiation was prevented by mutation. The failure of Clb6 to regulate reinitiation was due to rapid Clb6 proteolysis, since this toxic activity of Clb6 was lost when Clb6 was stabilized by mutation. Clb6-dependent toxicity is also relieved when early accumulation of mitotic cyclins is allowed to impose rereplication controls. Cell-cycle timing of rereplication control is crucial: sufficient rereplication block activity must be available as soon as firing begins. DNA rereplication induces DNA damage, and when rereplication controls are compromised, the DNA damage checkpoint factors Mre11 and Rad17 provide additional mechanisms that maintain viability and also prevent further rereplication, and this probably contributes to genome stability.

Plasma T790M Result Alters Treatment Options in a Previously T790 Wild-Type EGFR-Mutant Lung Cancer

Plasma T790M Result Alters Treatment Options in a Previously T790 Wild-Type EGFR-Mutant Lung Cancer Zofia Piotrowska, MD, MHS,* Benjamin Drapkin, MD, PhD, Jeffrey A. Engelman, MD, PhD, Rebecca J. Nagy, MS, LGC, Richard B. Lanman, MD, Lecia V. Sequist, MD, MPH Massachusetts General Hospital Cancer Center, Boston, Massachusetts Harvard Medical School, Boston, Massachusetts Guardant Health, Inc., Redwood City, California

Genomic and Functional Fidelity of Small Cell Lung Cancer Patient-Derived Xenografts

Small cell lung cancer (SCLC) patient-derived xenografts (PDX) can be generated from biopsies or circulating tumor cells (CTC), though scarcity of tissue and low efficiency of tumor growth have previously limited these approaches. Applying an established clinical-translational pipeline for tissue collection and an automated microfluidic platform for CTC enrichment, we generated 17 biopsy-derived PDXs and 17 CTC-derived PDXs in a 2-year timeframe, at 89% and 38% efficiency, respectively. Whole-exome sequencing showed that somatic alterations are stably maintained between patient tumors and PDXs. Early-passage PDXs maintain the genomic and transcriptional profiles of the founder PDX. In vivo treatment with etoposide and platinum (EP) in 30 PDX models demonstrated greater sensitivity in PDXs from EP-naïve patients, and resistance to EP corresponded to increased expression of a MYC gene signature. Finally, serial CTC-derived PDXs generated from an individual patient at multiple time points accurately recapitulated the evolving drug sensitivities of that patients disease. Collectively, this work highlights the translational potential of this strategy.Significance: Effective translational research utilizing SCLC PDX models requires both efficient generation of models from patients and fidelity of those models in representing patient tumor characteristics. We present approaches for efficient generation of PDXs from both biopsies and CTCs, and demonstrate that these models capture the mutational landscape and functional features of the donor tumors. Cancer Discov; 8(5); 600-15. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 517.

Abstract CT048: Phase 1/2 study of olaparib tablets and temozolomide in patients with small cell lung cancer (SCLC) following failure of prior chemotherapy

Background: SCLC is an aggressive high-grade neuroendocrine malignancy. While SCLC is often highly sensitive to first line platinum based chemotherapy, response rates to second line cytotoxic chemotherapy range from approximately 10-30%. Inhibition of poly(ADP-ribose) polymerase (PARP) by the PARP inhibitor olaparib (O) has shown activity in SCLC in preclinical studies, though it does not confer PFS or OS benefit in the maintenance setting as monotherapy (Ahmed et al., 2016). Temozolomide (T) is an alkylating agent with modest single agent activity (ORR 22% in the second line setting; Pietanza et al., 2012), and may synergize with PARP inhibitors. Methods: This ongoing phase 1/2 trial of combination O/T in adults with SCLC (NCT02446704) enrolled patients with histologically or cytologically confirmed incurable SCLC which had progressed following at least one prior platinum-based chemotherapy. O (tablet formulation) and T were administered orally on days 1-7 of 21-day cycles at escalating doses using a standard 3+3 design in the phase 1 portion, with a primary objective of determining the recommended phase 2 dose (RP2D). Response assessments were performed every 6 weeks. In parallel, patient derived xenografts (PDXs) were generated from biopsies and circulating tumor cells (CTCs) from a subset of patients both prior to O/T and at the time of acquired resistance. O/T activity was assessed in vivo in PDXs and in short term cultures. Results: In the phase 1 portion, 13 patients were enrolled to four escalating dose levels. No dose limiting toxicities or grade 4-5 toxicities were observed. The most common grade 3 related toxicities were neutropenia (38%), anemia (15%) and thrombocytopenia (15%). O 200 mg BID and T 75 mg/m2 QD was selected as the RP2D. There were 6 confirmed partial responses (ORR 46%) and responses were seen at all dose levels. The median progression free survival was 5.6 months and median duration of response was 3.4 months. In PDXs, the depth and duration of responses of models derived pre-O/T mirrored those of the corresponding patients. Most pre-treatment models were more sensitive to combination O/T than to either O or T alone. Models derived post-progression were highly resistant to O/T. Further testing to identify biomarkers of response and resistance is ongoing. Conclusions: O/T is well tolerated in patients with SCLC and shows promising clinical activity in a phase 1 study. PDXs derived from CTCs provide a powerful platform for performing co-clinical trials and modeling acquired resistance. Additional correlative studies will be presented. Citation Format: Anna F. Farago, Benjamin J. Drapkin, Allison Charles, Beow Yeap, Rebecca S. Heist, Christopher G. Azzoli, David M. Jackman, David A. Barbie, Edwin Choy, Lecia V. Sequist, Shyamala Maheswaran, Daniel A. Haber, Aaron N. Hata, Nicholas Dyson, Alice T. Shaw. Phase 1/2 study of olaparib tablets and temozolomide in patients with small cell lung cancer (SCLC) following failure of prior chemotherapy [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 CT048. doi:10.1158/1538-7445.AM2017-CT048

ATLANTIS: a Phase III study of lurbinectedin/doxorubicin versus topotecan or cyclophosphamide/doxorubicin/vincristine in patients with small-cell lung cancer who have failed one prior platinum-containing line

Lurbinectedin is an inhibitor of active transcription of protein-coding genes, causing DNA-break accumulation, apoptosis and modulation of the tumor microenvironment. Early-phase clinical trials indicate promising activity of lurbinectedin in small-cell lung cancer. Here, we describe the rationale and design of ATLANTIS (NCT02566993), an open-label, randomized, multicenter Phase III study to compare the efficacy of lurbinectedin and doxorubicin combination with standard-of-care chemotherapy, investigators choice of cyclophosphamide/doxorubicin/vincristine or topotecan, in patients with small-cell lung cancer that has progressed following one line of platinum-based chemotherapy. Patients are randomized in a 1:1 ratio. The primary end point is overall survival and key secondary end points include progression-free survival, best tumor response and duration of response, each assessed by independent review committee.