Efficacy Of DNA-damaging Agents Research Articles

A Two-Part Study Evaluating the Combination of Tazemetostat and CPX-351 (Part 1) and Palbociclib with CPX-351 (Part 2) for the Treatment of Relapsed or Refractory Acute Myeloid Leukemia

BACKGROUND AND SIGNIFICANCE Acute myeloid leukemia (AML) is an aggressive cancer of early hematopoietic progenitor cells with only 25-30% of patients (pts) surviving 5 years from diagnosis. For fit pts, conventional chemotherapy with DNA-damaging agents remains the mainstay of treatment. Conventional chemotherapeutic agents cause DNA-damage by targeting cells during DNA replication. Chromatin structure is highly influential in the efficacy of DNA-damaging agents, as areas of compacted chromatin are more resistant to DNA-damaging effects. Areas of DNA marked with histone 3 lysine 27 trimethylation (H3K27me3) are transcriptionally repressed and exhibit reduced accessibility to DNA-damaging agents. Enhancer of Zeste Homolog 2 (EZH2) serves as the catalytic subunit of the polycomb repressive complex 2 which methylates H2K27 to produce a di- or trimethylated state (H3K27me2/3). In preclinical studies, treatment of AML cell lines with the combination of an EZH2 inhibitor (EZH2i) and doxorubicin resulted in an increase in DNA damage and apoptosis compared to those treated with doxorubicin alone. Further, a significant decrease in leukemia burden was seen in mice injected with THP-1 (AML) cells and treated with an EZH2i and doxorubicin and cytarabine compared to doxorubicin and cytarabine alone. This effect was further enhanced by pre-treatment of AML cells with palbociclib (PALB), a CDK4/6 inhibitor, by significantly increasing the number of cells in S phase when exposed to the EZH2i and doxorubicin combination, resulting in an increase in DNA damage and apoptosis compared to cells that were not pre-treated with PALB. These findings were reproduced in in vivo studies with mice injected with the primary AML cells (Porazzi P, et al. Cancer Res., 2021). STUDY DESIGN AND METHODS We will perform a 2-part phase I dose escalation study utilizing EZH2i tazemetostat (TAZ) in combination with CPX-351 induction chemotherapy and of PALB pre-treatment followed by CPX-351 in pts with relapsed or refractory (R/R) AML (NCT05627232). All pts will receive CPX-351 at standard dose on days 1, 3 and 5. Part 1 evaluate the addition of TAZ to CPX-351 via traditional 3+3 design. Pts will be treated with TAZ on days -1 to 6 at a starting dose of 600 mg twice daily to open chromatin concurrent with CPX-351 administration. Part 2 evaluate the pre-treatment with PALB via traditional 3+3 design. Pts will be treated with PALB starting dose of 100 mg daily on days -3 to -1 to induce cell cycle G1. PALB will be held on day 1 to allow cells to then enter S phase prior to administration of CPX-351. ( Figure 1). The study will enroll pts with R/R AML treated at Sidney Kimmel Cancer Center. Pts must be ≥18 years of age with R/R AML (non-acute promyelocytic leukemia) and have received at least 1 prior line of therapy and have adequate performance status and organ function. Pts whose participation would result in a total cumulative dose of daunorubicin greater than 550 mg/m 2 (450 mg/m 2 if they previously received mediastinal radiation) and those with active central nervous system involvement of AML, unstable cardiac disease or other severe concurrent disease will be excluded. The primary endpoints of Parts 1 and 2 will be to determine the maximum tolerated dose of TAZ and of PALB in combination with CPX-351, respectively. Secondary endpoints for Parts 1 and 2 include preliminary assessment of efficacy. Correlative studies to evaluate the on-target effect of TAZ on H3K27me3 and DNA-damage and of PALB on cell cycling will be performed at protocol-specified time points. If these two 2-drug combinations prove to be safe, we will amend the protocol to add Part 3 which will evaluate the safety of the 3-drug combination of pre-treatment with PALB followed by co-treatment with TAZ and CPX-351.

Reduced Tumorigenicity of Mouse ES Cells and the Augmented Anti-Tumor Therapeutic Effects under Parg Deficiency.

PolyADP-ribosylation is a post-translational modification of proteins, and poly(ADP-ribose) (PAR) polymerase (PARP) family proteins synthesize PAR using NAD as a substrate. Poly(ADP-ribose) glycohydrolase (PARG) functions as the main enzyme for the degradation of PAR. In this study, we investigated the effects of Parg deficiency on tumorigenesis and therapeutic efficacy of DNA damaging agents, using mouse ES cell-derived tumor models. To examine the effects of Parg deficiency on tumorigenesis, Parg+/+ and Parg−/− ES cells were subcutaneously injected into nude mice. The results showed that Parg deficiency delays early onset of tumorigenesis from ES cells. All the tumors were phenotypically similar to teratocarcinoma and microscopic findings indicated that differentiation spectrum was similar between the Parg genotypes. The augmented anti-tumor therapeutic effects of X-irradiation were observed under Parg deficiency. These results suggest that Parg deficiency suppresses early stages of tumorigenesis and that Parg inhibition, in combination with DNA damaging agents, may efficiently control tumor growth in particular types of germ cell tumors.

Abstract P3-10-08: A phase II study of cisplatin and the Wee1 inhibitor adavosertib in metastatic triple-negative breast cancer (mTNBC)

Abstract Introduction: mTNBC progresses rapidly on first-line chemotherapy. For instance, in the TNT trial, median progression-free survival (PFS) was 3 months on first-line carboplatin. Selective inhibition of Wee1, a negative regulator of the G2 cell cycle checkpoint, may enhance the efficacy of DNA-damaging agents by reducing DNA damage repair. We report the results of the first phase II study assessing the efficacy of the Wee1 inhibitor adavosertib (AZD-1775) with cisplatin in mTNBC. Methods: mTNBC patients (pts) with 0-1 prior lines of chemotherapy in the metastatic setting were eligible. Pts received cisplatin 75 mg/m2 IV followed by combination therapy 21 d later with cisplatin plus adavosertib 200 mg oral twice daily x 5 doses over 2.5 d every 21 d. Tumor biopsies were mandatory for patients with safely accessible tumors, and occurred 5-48 h after monotherapy and 5-8 h after the last adavosertib dose in the first combination therapy cycle. The study used a Simon optimal two-stage design that had 90% power to detect the difference between null (20%) and alternative (40%) response rates with a one-sided type I error of 0.1. The primary endpoint was objective response rate (ORR): an ORR > 30% would identify the regimen as worthy of further study. Key secondary endpoints were PFS and overall survival (OS). Exploratory analyses evaluated the association of response, defined as clinical benefit rate (CBR) > 6 months (CR + PR + SD > 6 mos), with transcriptional and immunostaining profiles of on-treatment tumors (n = 16), as well as with targeted panel genomic alterations in archival tissue. Tumor immune cell composition was assessed by CIBERSORT. Results: 34 pts initiated protocol therapy; median age was 56 yrs, 2 (6%) pts had known BRCA2 mutations, and 14 (41%) had 1 prior line of chemotherapy. Median follow-up was 13 mos. ORR was 29% (3 CR + 7 PR), median PFS 4.9 months (95% CI, 2.3-5.8 mos), CBR 35%, and preliminary OS 14.0 mos (95% CI, 11.8-21.8 mos). All-cause AEs occurred in 100% of pts (G3-4, 56%; most commonly diarrhea, 21%), including one death due to sepsis possibly related to study therapy. The primary tumor of the longest responding patient had a possible biallelic loss-of-function alteration (missense mutation and loss of heterozygosity) in the homologous recombination-related gene FANCM. In Hallmark gene set enrichment analyses of 26 on-treatment biopsies across 16 pts, responding tumors (n = 4) demonstrated enriched expression of immune response gene sets (allograft rejection FDR q < 0.001, IL2-STAT5 signaling FDR q = 0.001, inflammatory response FDR q = 0.003), while non-responding tumors (n = 12) showed enrichment of cell cycle gene sets (E2F targets FDR q < 0.001, G2M checkpoint FDR q < 0.001). In the same on-treatment biopsies, responding tumors (n = 4) had higher tumor infiltrating lymphocytes (46% v. 29%, Mann-Whitney p = 0.04) and lower M0 macrophages (3% v. 17%, Mann-Whitney p = 0.04) than non-responding tumors (n =12). Updated data, including OS and tissue immunostaining results, will be presented. Conclusions: Among mTNBC pts, the combination of adavosertib and cisplatin was associated with a 29% ORR, failing to meet the pre-determined ORR of > 30% required to support further investigation. Responses were associated with immune-related gene expression and TILs detected by RNA sequencing. Citation Format: Tanya E. Keenan, Zhenying Tan-Wasielewski, Lorenzo Trippa, Bose Kochupurakkal, Jennifer L. Guerriero, Rie K. Tahara, Grace Winship, Wafa Osmani, Chelsea Andrews, Jake R. Conway, Meng X He, Ricardo Pastorello, Adam Tracy, Robert E. Godin, Beth A. Overmoyer, Eric P. Winer, Elizabeth A. Mittendorf, Geoffrey I Shapiro, Eliezer M Van Allen, Sara M Tolaney. A phase II study of cisplatin and the Wee1 inhibitor adavosertib in metastatic triple-negative breast cancer (mTNBC) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-10-08.

Abstract 2876: Targeting VCP to enhance pancreatic cancer response to DNA damaging agents

Abstract Dysregulation of DNA damage response and repair (DDR) is common in pancreatic and other cancers, and impairs their sensitivity to DNA damaging agents. Targeting DDR mediators is therefore an attractive strategy to improve response to DNA damaging agents. We have identified the AAA ATPase VCP as a lead candidate for this via siRNA screening. VCP-chemotherapy synergistic combinations have not as yet been explored in solid tumours. To investigate the mechanism by which VCP modulates response to cisplatin-induced DNA damage, the effect of VCP functionality loss in decreasing cell viability was validated using two pharmacological VCP inhibitors, NMS-873 and CB-5083, in a panel of pancreatic cancer cell lines. Effects on ovarian cancer cell lines were also assessed for comparison, and IC50 values (72 hours) were found to range from 0.03 - 1.4μM. Greater sensitivity to both inhibitors corresponded with accumulation of K48-ubiquitinated protein levels, and increased expression of the unfolded protein response-related genes BiP, DDIT3, and the spliced form of XBP1. NMS-873 (10nM) or CB-5083 (50nM) were also found to increase cisplatin sensitivity (decreased IC50 values, 48 hours) in MIA-PaCa-2, SKOV-3, PEO1, and PEO4 cell lines, but not in AsPC-1 or PANC-1 cells. To investigate if cisplatin sensitization relates to changes in DDR activity, DNA double strand break repair (DSB) assays, using GFP-reporter constructs, were carried out but lower efficiency of homologous recombination (HR) and non-homologous end-joining pathways were found in both PANC-1 and SKOV-3 cells following VCP inhibition. Consistent with decreased HR activity, VCP inhibition reduced numbers of cisplatin-induced Rad51 foci in both cell lines. Unexpectedly, this was associated with increased proteasomal degradation of Rad51, as indicated by western blotting. Phosphorylation of VCP at serine 784 was detected following cisplatin treatment, and interestingly was found to be reduced following inhibition of DNA-PKcs, one of the main PI3K-related kinases (PIKKs) which directs DSB repair. To explore the role of this modification, a phospho-deficient VCP, with mutations in 4 predicted PIKK-targeted sites (including serine 784), was generated. A significant increase in apoptosis, as measured by Caspase-Glo®3/7 assay, was observed in cisplatin-treated SKOV-3 cells, as an initial model system, expressing this phospho-deficient VCP. Studies are underway to further elucidate the importance of each individual phosphorylation site on cisplatin response and DSB repair. Our findings suggest VCP could be a potential therapeutic target for pancreatic and other cancers. Confirming how VCP regulates DSB repair will allow development of novel strategies to enhance the efficacy of DNA damaging agents in cancer. Citation Format: Yuliana Astuti, Charlene Lam, Harpreet S. Wasan, Elaina N. Maginn. Targeting VCP to enhance pancreatic cancer response to DNA damaging agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2876.

Abstract 2829: Targeting the DNA damage response and DNA-PK signaling via small molecule Ku inhibitors

Abstract Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin-top:0in; mso-para-margin-right:0in; mso-para-margin-bottom:10.0pt; mso-para-margin-left:0in; line-height:115%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin;} The DNA dependent protein kinase (DNA-PK) is a validated target for cancer therapeutics that drives the DNA damage response (DDR) and plays a critical role as a primary sensor in the non-homologous end joining (NHEJ) DNA double strand break (DSB) repair pathway. Various anti-cancer therapeutic strategies mediate their cytotoxic effects by inducing DSBs, including ionizing radiation (IR), and clinical outcomes are directly related to the repair of DNA damage. Modulating the pathway responsible for repairing DSBs will have a profound impact on the efficacy of DNA damaging agents in the clinic. To date, development of inhibitors for DNA-PK has focused on targeting the active site with ATP mimetics. We have taken the novel and innovative approach to inhibiting DNA-PK via blocking the Ku 70/80 heterodimer interaction with DNA, a necessary step in DNA-PK activation. Exploiting this unique mechanism of kinase activation, we have identified a series of highly potent and specific DNA-PK inhibitors that impart their inhibitory activity via disruption of the binding of Ku protein to DNA ends. This novel approach affords significant advantages to current approaches in kinase inhibition. Novel derivatives of our initial hit inhibit DNA-PK catalytic activity at nanomolar concentrations and potentiate cellular sensitivity to DSB-inducing agents like etoposide and bleomycin. Data demonstrate that the cellular effects observed are a function of Ku inhibition and that this novel class of DNA-PK inhibitors can be further developed as anti-cancer therapeutics that can be used as an adjuvant to, or concomitant with radiotherapy and other cancer therapies that induce DNA damage. Citation Format: John J. Turchi, Navnath S. Gavande, Pamela S. VanderVere-Carozza, Tyler Vernon, Katherine S. Pawelczak. Targeting the DNA damage response and DNA-PK signaling via small molecule Ku inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2829.

Adventures in Scaffold Morphing: Discovery of Fused Ring Heterocyclic Checkpoint Kinase 1 (CHK1) Inhibitors.

Checkpoint kinase 1 (CHK1) inhibitors are potential cancer therapeutics that can be utilized for enhancing the efficacy of DNA damaging agents. Multiple small molecule CHK1 inhibitors from different chemical scaffolds have been developed and evaluated in clinical trials in combination with chemotherapeutics and radiation treatment. Scaffold morphing of thiophene carboxamide ureas (TCUs), such as AZD7762 (1) and a related series of triazoloquinolines (TZQs), led to the identification of fused-ring bicyclic CHK1 inhibitors, 7-carboxamide thienopyridines (7-CTPs), and 7-carboxamide indoles. X-ray crystal structures reveal a key intramolecular noncovalent sulfur-oxygen interaction in aligning the hinge-binding carboxamide group to the thienopyridine core in a coplanar fashion. An intramolecular hydrogen bond to an indole NH was also effective in locking the carboxamide in the preferred bound conformation to CHK1. Optimization on the 7-CTP series resulted in the identification of lead compound 44, which displayed respectable drug-like properties and good in vitro and in vivo potency.

Abstract LB-A11: Targeting DNA-PK and the DNA damage response via small molecule Ku inhibitors

Abstract The DNA dependent protein kinase (DNA-PK) is a validated target for cancer therapeutics that drives the DNA damage response (DDR) and plays a critical role as a primary sensor in the non-homologous end joining (NHEJ) DNA double strand break (DSB) repair pathway. Various anti-cancer therapeutic strategies mediate their cytotoxic effects by inducing DSBs, including ionizing radiation (IR), and clinical outcomes are directly related to the repair of DNA damage. Modulating the pathway responsible for repairing DSBs will have a profound impact on the efficacy of DNA damaging agents in the clinic. To date, development of inhibitors for DNA-PK has focused on targeting the active site with ATP mimetics. We have taken the novel and innovative approach to inhibiting DNA-PK via blocking the Ku 70/80 heterodimer interaction with DNA, a necessary step in DNA-PK activation. Exploiting this unique mechanism of kinase activation, we have identified a series of highly potent and specific DNA-PK inhibitors that impart their inhibitory activity via disruption of the binding of Ku protein to DNA ends. This novel approach affords significant advantages to current approaches in kinase inhibition. Novel derivatives of our initial hit inhibit DNA-PK catalytic activity at nanomolar concentrations and potentiate cellular sensitivity to DSB-inducing agents like etoposide and bleomycin. Data demonstrate that the cellular effects observed are a function of Ku inhibition and that this novel class of DNA-PK inhibitors can be further developed as anti-cancer therapeutics that can be used as an adjuvant to, or concomitant with radiotherapy and other cancer therapies that induce DNA damage. Citation Format: John J. Turchi, Navnath Gavande, Pamela S. VanderVere-Carozza, Tyler Vernon, Katherine S. Pawelczak. Targeting DNA-PK and the DNA damage response via small molecule Ku inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-A11.

EXD2 - a new player joins the DSB resection team

EXD2 - a new player joins the DSB resection team

HuR Posttranscriptionally Regulates WEE1: Implications for the DNA Damage Response in Pancreatic Cancer Cells

HuR (ELAV1), an RNA-binding protein abundant in cancer cells, primarily resides in the nucleus, but under specific stress (e.g., gemcitabine), HuR translocates to the cytoplasm in which it tightly modulates the expression of mRNA survival cargo. Here, we demonstrate for the first time that stressing pancreatic ductal adenocarcinoma (PDA) cells by treatment with DNA-damaging anticancer agents (mitomycin C, oxaliplatin, cisplatin, carboplatin, and a PARP inhibitor) results in HuR's translocation from the nucleus to the cytoplasm. Importantly, silencing HuR in PDA cells sensitized the cells to these agents, whereas overexpressing HuR caused resistance. HuR's role in the efficacy of DNA-damaging agents in PDA cells was, in part, attributed to the acute upregulation of WEE1 by HuR. WEE1, a mitotic inhibitor kinase, regulates the DNA damage repair pathway, and therapeutic inhibition of WEE1 in combination with chemotherapy is currently in early phase trials for the treatment of cancer. We validate WEE1 as a HuR target in vitro and in vivo by demonstrating (i) direct binding of HuR to WEE1's mRNA (a discrete 56-bp region residing in the 3' untranslated region) and (ii) HuR siRNA silencing and overexpression directly affects the protein levels of WEE1, especially after DNA damage. HuR's positive regulation of WEE1 increases γ-H2AX levels, induces Cdk1 phosphorylation, and promotes cell-cycle arrest at the G2-M transition. We describe a novel mechanism that PDA cells use to protect against DNA damage in which HuR posttranscriptionally regulates the expression and downstream function of WEE1 upon exposure to DNA-damaging agents.

Abstract LB-299: Comprehensive preclinical evaluation of VE-822, the first ATR-targeted drug candidate: a novel approach to transforming the efficacy of DNA damaging agents.

Abstract DNA damaging agents have been the cornerstone of cancer therapy for decades yet they provide only modest benefit for most patients. For example, standard of care for patients with non-small cell lung cancer (NSCLC) is dominated by the use of platinating drugs and ionizing radiation (IR), however outcome remains very poor with 5-year survival rates of <15% for patients that present with advanced disease. Such poor responses to DNA damaging treatment reflects, in part, the efficient repair of DNA damage via a complex signaling and repair network known as the DNA damage response (DDR). The DDR detects double strand breaks and replication stress, the most lethal forms of DNA damage, and acts to enforce checkpoints to halt cell cycle progression, and to stimulate repair. Key regulators of the DDR are the phosphoinositol 3-kinase-like serine/threonine protein kinase (PIKK) family members ATR and ATM. Recent pre-clinical data has suggested that a reliance on ATR for survival from DNA damage may be a common feature of cancer. This can arise either as a consequence of high replicative stress, for example from expression of certain oncogenes, from a hypoxic microenvironment, or from defects elsewhere in DNA damage surveillance and repair pathways. Most notably inhibition of ATR has been shown to be synthetic lethal with loss of the ATM-p53 pathway. In NSCLC defective ATM signaling, from loss of ATM expression or from defects in p53 has been reported in about 50% of tumors. Here we describe the comprehensive in vitro and in vivo profile for VE-822 a novel highly potent and selective inhibitor of ATR. VE-822 potently inhibits ATR in biochemical assays with Ki <0.3nM and in cell assays with IC50 of 20nM. Against a large panel of NSCLC lines, low concentrations of VE-822 sensitized many lines to the cytotoxic effects of multiple DNA damaging agents; for example >90% of lines showed >3-fold shifts in IC50 for cisplatin in the presence of VE-822, with ~50% of lines showing >10-fold increases in cisplatin cytotoxicity. In contrast normal cells tolerate inhibition of ATR. In a panel of mouse xenograft models, derived from various primary human NSCLC tumor tissues, oral or IV administration of VE-822 strongly sensitized tumors to cisplatin treatment. In many cases, combinations including VE-822 led to tumor regression or extensive tumor growth delay. Inhibition of ATR activity and accumulation of DNA damage by VE-822 was observed coincident with efficacy. When administered alone or in combination with cisplatin VE-822 was well tolerated in mice at doses that block ATR activity. These data support the potential for ATR inhibitors to substantially increase the efficacy of standard-of-care agents in diseases such as NSCLC. Citation Format: Diane Boucher, Peter Charlton, Jean-Damien Charrier, Brinley Furey, Yong Gu, Amy Hall, Brian Hare, Howard Li, Sean Milton, Cheryl Murphy, Philip Reaper, Darin Takemoto, Taturo Udagawa, Yuxin Wang, Mark Wood, John Pollard. Comprehensive preclinical evaluation of VE-822, the first ATR-targeted drug candidate: a novel approach to transforming the efficacy of DNA damaging agents. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-299. doi:10.1158/1538-7445.AM2013-LB-299

Abstract PD09-02: BRCA1 insufficiency is predictive of superior survival in patients with triple negative breast cancer treated with platinum based chemotherapy

Abstract Introduction: Triple negative breast cancer (TNBC) and BRCA1-associated breast cancers share many histopathologic and molecular features. BRCA1 plays a crucial role in HR-dependent DNA repair and BRCA1-deficient cells are particularly susceptible to the DNA damaging agents like platinums. Increasing evidence suggests that in addition to germline BRCA defects, other mechanisms (like epigenetic BRCA1 silencing) can lead to BRCA1 insufficiency in TNBC. However, the impact of BRCA1 insufficiency on the efficacy of DNA damaging agents in TNBC is not known. Aim: To investigate the impact of BRCA1 insufficiency on relapse-free survival (RFS) and overall survival (OS) in patients with stage II-III TNBC treated with neoadjuvant platinum-based chemotherapy. BRCA1 insufficiency (BRCA1insuf) state was defined as presence of germline BRCA1/2 mutation or BRCA1 promoter methylation (PM) and/or low BRCA1 expression (lowest quartile). Methods: Thirty patients with stage II/III TNBC received neoadjuvant chemotherapy (6 cycles of Carboplatin AUC 6, Docetaxel 75mg/m2 and Erlotinib 150 mg PO) on a phase II trial between 8/2007–6/2010. All but one patient underwent comprehensive BRCA analysis (Myriad Genetic Laboratories). Pre-treatment tumor specimens were used for evaluation of BRCA1 PM and expression. Genomic DNA was isolated from FFPE samples, bisulfite converted and then subjected to methylation-specific PCR (MSP). RNA was isolated, reverse transcribed to cDNA and assayed by quantitative real-time PCR (qRT-PCR) for determination of BRCA1 mRNA transcript levels. RFS and OS were estimated according to the Kaplan-Meier method and compared among groups with log-rank statistic. Cox proportional hazards models were fit to determine the association of BRCA1insuf with the risk of death after adjustment for other characteristics. Results: Median age: 51yrs, African American: 20%, Median tumor size: 3.3 cm, LN positive: 40%. Six of 30 patients (20%) harbored germline BRCA mutation (4 BRCA1, 2 BRCA2). Baseline tumor specimen was available for 26/30 patients. BRCA1 MSP was successful in 92% and BRCA1 qRT-PCR was successful in 84% of specimens. BRCA1 PM and low BRCA1 expression was present in 30% and 15% of subjects, respectively. There was evidence of BRCA1insuf in 53% (16/30) of subjects. At a median time from diagnosis of 42 months (range, 23–59 months) there have been 9(30%) recurrences and 7(23%) deaths. On univariate analysis node negativity, lower stage and presence of BRCA1insuf were associated with better OS. At the median follow up, RFS is 81% for patients with BRCA1insuf versus 54% for patients without BRCA1insuf (p = 0.16); OS is 83% for patients with BRCA1insuf versus 46% for patients without BRCA1insuf (p = 0.021). After adjustment for clinical variables patients with BRCA1insuf had a significantly better OS compared to patients without BRCA1insuf (p = 0.036). Conclusions: Germline BRCA testing plus tissue BRCA1 PM/expression can be used to identify a BRCA1insuf sub-population within TNBC demonstrating a favorable outcome with platinum treatment. This BRCA1insuf criteria can be easily used to select TNBC patients likely to benefit from DNA damaging agents like platinums and PARP inhibitors. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD09-02.

Abstract 4694: The small molecule CHK1/CHK2 inhibitor PF-0477736 (Pfizer) demonstrates single agent activity in diffuse large B-cell lymphoma

Abstract Few data are available on the role of CHK inhibitors in Diffuse Large B cell Lymphoma (DLBCL). The checkpoint kinases 1 (CHK1) and 2 (CHK2) are serine-threonine kinases involved in the DNA damage response pathway. CHK inhibition enhances the efficacy of DNA damaging agents in a variety of tumors, including p53 deficient cells, that rely on the G2/M checkpoint, having a dysfunctional G1 checkpoint. DLBCL with dysfunctional p53 axis (harboring p53 mutations and/or CDKN2A loss) have been recently shown to have a dismal outcome. In this study we report the activity profile of the CHK1/2 inhibitor PF-0477736 (Pfizer) in a panel of B cell lymphoma cell lines and primary cells, and explore its mechanisms of action. Three Germinal center B cell (GCB) DLBCL derived cell lines (SUDHL-4, SHDHL-6, BJAB), 3 Activated B cell (ABC) DLBCL (HBL-1, U2932, TMD8), 2 mantle cell lymphoma (Mino, SP-53), and the Hodgkin Lymphoma cell line KM-H2 were first screened for p53 and CDKN2A mutations and deletions. P53 mutations were detected in the following cell lines: HBL-1, U2932, SUDHL-6, BJAB, Mino, SP-53. TMD8 was p53 wild-type but with an homozygous deletion of CDKN2A. Of note SUDHL-4 and KM-H2 were p53 wild type, with no deletion of CDKN2A. To assess the effect of PF-0477736 on cell proliferation, cells were incubated with increasing concentrations of PF-0477736 (from 5 to 2000 nM) for 24, 48 and 72 hours (hrs), and cell viability assessed by WST-1 assay (Roche). A significant growth inhibition was evident after 48 hrs, in all cell lines, excluding SUDHL-4 and KM-H2 that were resistant (IC50 8300 and 6800 nM at 48 hrs, respectively). The BJAB cell line showed the highest sensitivity (IC50 10 nM at 24 hrs). The IC50 ranged from 140 to 230 nM at 48 hrs in the other sensitive cell lines. Using Annexin V- propidium iodide staining, we found that PF-0477736 25-500 nM induced cell death by apoptosis in a time and dose dependent manner. Of note PF-0477736 100-1000 nM demonstrated activity also in primary DLBCL cells. We found no correlations between baseline levels of CHK1/2 activation and outcome. In the sensitive cell lines inhibition of the downstream target CDC25c ser216 phosphorylation coupled with a marked increase in levels of the DNA damage marker γH2AX was observed by western blot as soon as after 24 hrs of incubation with concentrations equal to the IC50 (25 - 250 nM). PF-0477736 at the dose of 50-100 nM synergistically enhanced the efficacy of Doxorubicin (0.1 to 1 μM) at 24 hrs. These data suggest that PF-0477736 has single agent activity and synergizes with chemotherapy in DLBCL. The drug shows high single agent activity in the subset of DLBCL with genomic lesions of the p53 pathway, that are resistant to conventional chemotherapy and associated with dismal outcome, providing the rationale for further clinical investigation of PF-0477736 in DLBCL alone or in combination with chemotherapy. PF-0477736 was provided by Pfizer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4694. doi:1538-7445.AM2012-4694

Inhibition of poly(ADP‐ribose) polymerase (PARP) and ataxia telangiectasia mutated (ATM) on the chemosensitivity of mantle cell lymphoma to agents that induce DNA strand breaks

There is a high incidence of genomic aberration of ataxia telangiectasia mutated (ATM) and genes encoding proteins involved in the ATM pathway in mantle cell lymphoma (MCL). It has been shown that poly(ADP-ribose) polymerase inhibitor (PARPi) strongly enhances the cytotoxicity of agents, causing single-strand DNA breaks in cells with impaired homologous recombination repair. Here, we show that PARPi AG14361 potentiates the cytotoxicity induced by topotecan treatment in MCL cell lines, which was not dependent on either TP53 or CHEK2 status. Inhibition and/or knockdown of ATM and BRCA2 did not potentiate the cytotoxic effect of treatment with PARPi and topotecan. With loss of function of ATM, other kinases can still mediate activation of ATM substrates as demonstrated by continued phosphorylation of CHEK2 (Thr-68), although attenuated and delayed. These results suggest that PARPi may enhance the therapeutic efficacy of DNA damaging agents on MCL through TP53-independent mechanisms without requiring the inhibition of either ATM or BRCA2.

The Small Molecule CHK1/CHK2 Inhibitor PF-0477736 (Pfizer) Demonstrates Single Agent Activity and Synergizes with Chemotherapy in Diffuse Large B-Cell Lymphoma

Abstract 2732The checkpoint kinases 1 (CHK1) and 2 (CHK2) are serine-threonine kinases involved in the signal transduction mechanims of the DNA damage response pathway. Once activated by upstream kinases [Ataxia-Telangiectasia mutated (ATM) and Ataxia-Telangiectasia and Rad3-related (ATR) kinases] following DNA damage, they phosphorylate downstream targets such as CDC25 phosphatases and p53, promoting G2/M cell cycle arrest, in order to facilitate DNA repair. Furthermore is now clear that the efficacy of conventional DNA-damaging anticancer drugs is limited by the activity of these protective cell cycle checkpoints. The tumor suppressor p53 is activated in normal cells following extensive DNA damage and promotes G1 cell cycle arrest and apoptosis. Cells lacking p53 activity are more resistant to genotoxic agents. It has been shown that CHK inhibition enhances the efficacy of DNA damaging agents in a variety of tumors, by inhibiting the response to DNA damage, preferentially in p53 deficient cells, that rely on the G2/M checkpoint, having a dysfunctional G1 checkpoint. DLBCL harboring p53 mutations and/or CDKN2A loss have been recently shown to have a dismal outcome, being refractory to conventional antracyclin-based chemotherapy. Few data are available on the role of CHK inhibitors in Diffuse Large B cell Lymphoma (DLBCL). In this study we report the activity profile of the CHK1/2 inhibitor PF-0477736 (Pfizer) in a large panel of B cell lymphoma cell lines, and explore its mechanisms of action. Nine cell lines were used for in vitro viability assays: 3 Germinal center (GCB) Diffuse Large B-cell lymphoma (DLBCL) derived cell lines (SUDHL-4, SHDHL-6, BJAB), 3 Activated B cell (ABC) DLBCL (HBL-1, U2932, TMD8), 2 mantle cell lymphoma (Mino, SP-53), and the Hodgkin Lymphoma cell line KM-H2. All the cell lines were screened for p53 and CDKN2A mutations and deletions. P53 mutations were detected in the following cell lines: HBL-1, U2932, SUDHL-6, BJAB, Mino, SP-53. TMD8 was p53 wild-type but an homozygous deletion of CDKN2A was detected. Of note SUDHL-4 and KM-H2 were p53 wild type, with no deletion of CDKN2A. To assess the effect of PF-0477736 on cell proliferation, cells were first incubated with increasing concentrations of PF-0477736 (from 5 to 2000 nM) for 24, 48 and 72 hours (hrs), and cell viability assessed by WST-1 assay (Roche). A significant growth inhibition was evident after 48 hrs of incubation, in all cell lines, excluding SUDHL-4 and KM-H2 that were resistant (IC50 8300 and 6800 nM at 48 hrs, respectively). The BJAB cell line showed the highest sensitivity, with a decrease in cell viability close to 50% following incubation with PF-0477736 10nM for 24 hours. The IC50 ranged from 140 to 230 nM at 48 hrs in the other sensitive cell lines. Using Annexin V- propidium iodide staining, we found that PF-0477736 250–500 nM induced cell death by apoptosis in a time and dose dependent manner after 24 and 48 hours of incubation. Lower concentrations of PF-0477736 (25–50 nM) promoted a statistically significant increase in cell death only in the BJAB cells. For functional studies we characterized the two most sensitive cell lines (BJAB and U2932) and the two resistant cell lines (SUDHL-4 and KM-H2). Inhibition of cdc25c ser216 phosphorylation was observed by western blot as soon as after 24 hrs of incubation with concentrations equal to the IC50 (25–250 nM). A marked increase in levels of the DNA damage marker γH2AX, was detected in the BJAB, U2932, SUDHL-4 cell lines after 24 hrs. KM-H2 did not show any increase of γH2AX following treatment. All the cell lines demonstrated baseline CHK1 activation but there was no correlation with outcome. Interestingly levels of baseline pcdc25c ser216 were higher in the sensitive BJAB and U2932 cells. PF-0477736 at the fixed dose of 50 nM synergistically enhanced the efficacy of Doxorubicin (0.1 to 1 μM) in the BJAB and U2932 cells at 24 hrs.These data suggest that PF-0477736 has single agent activity and synergizes with chemotherapy in DLBCL. The integrity of the p53 axis seems to be the major determinant of efficacy of PF-0477736. The drug shows high single agent activity in the subset of DLBCL with genomic lesions of the p53 pathway, that are resistant to conventional chemotherapy and associated with dismal outcome. Our study provides the rationale for further clinical investigation of PF-0477736 in DLBCL alone or in combination with chemotherapy.PF-0477736 was provided by Pfizer. Disclosures:No relevant conflicts of interest to declare.

Abstract 3931: Elevated levels of poly(ADP-ribose) lead to enhanced efficacy of DNA-damaging chemotherapeutic agents

Abstract The synthesis of poly(ADP-ribose) (PAR) is an immediate response to DNA damage. We reported previously that the failure to degrade PAR by inhibiting PAR glycohydrolase (PARG) leads to hypersensitivity to DNA damage. To investigate the ability of PARG inhibition to increase the effectiveness of DNA-damaging agents, we analyzed chromatin structure, DNA damage and repair, and cell death in PARG null trophoblast stem cells. PARG null cells displayed high levels of PAR-modified histones H1 and H2B, suggesting chromatin structural change. Chromatin decondensation was observed in PARG null cells by transmission electron microscopy. This structural change led to increased access of the DNA-modifying agent acridine orange and micrococcal nuclease. Analysis by Comet assay demonstrated an extraordinary amount of DNA damage induced by sublethal amounts of the DNA alkylating agent N-methyl-N’-nitro-N-nitrosoguanidine that was subsequently not repaired in PARG null cells. To determine if this chromatin decondensation might lead to enhanced access of DNA to currently utilized DNA-modifying chemotherapeutic agents, PARG null cells were treated with cisplatin and cyclophosphamide. Each induced 2 to 4 folds greater amounts of cell death in PARG null cells. Taken together, the results show that PARG inhibition enhances the efficacy of DNA-damaging agents by inducing a greater amount of DNA damage, a decrease in DNA repair, and an increase in cell death. The results therefore suggest that targeting PARG will enhance the effectiveness of DNA-modifying anti-cancer agents. This research was supported by funding from the American Cancer Society and the Pharmaceutical Research Manufacturers of America. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3931.

Abstract A251: MK-1775, a potent Wee1 inhibitor, synergizes with gemcitabine to achieve tumor regressions in pancreatic tumor xenografts

Abstract Background and Aim: Treatment efficacy of DNA damaging agents is determined not only by the amount of therapy-induced DNA damage but also by the capacity of tumor cells to repair the damaged DNA. When cells are treated with DNA damaging agents, both G1 and G2/M cell cycle checkpoints are activated, leading to cell cycle arrest, thus providing time for the cell to repair the damage and to evade apoptosis before resuming cell cycle. Tumor cells can exploit these repair mechanisms in response to DNA damaging chemotherapeutics, rendering tumors refractory to therapy. Since G1 checkpoint is frequently compromised due to loss-of-function mutation in p53 gene in approximately half of cancers, G2/M checkpoint play a pivotal role in preventing the programmed cell death in p53 null tumors. Wee1 kinase, which acts as a critical driver of G2-M cell cycle progression, plays a critical role in maintaining G2 arrest through its inhibitory phosphorylation of cdc2. G2 checkpoint abrogation by Wee1 inhibition may therefore sensitize p53 deficient tumor cells to anti-cancer agents. MK-1775, a potent inhibitor of Wee1 tyrosine kinase, is currently being evaluated in several phase I clinical trials. Here, we investigated the efficacy and pharmacodynamic effects of MK-1775 in both monotherapy and in combination with gemcitabine in pancreatic cancer xenografts with p53 null/intact status. Methods: Fresh pancreatic tumor pieces obtained from patients at the time of surgery in Johns Hopkins Hospital were implanted (s.c) in female athymic mice and grafted tumors were propagated as a live PancXenoBank. Ten patient xenografts (7 with p53 mutated and 3 with p53 wild type) were allowed to grow separately on both flanks of mouse. When tumor reached a volume of ∼200 mm3, mice were individually identified and randomly assigned to treatment groups, with 5–6 mice (8–10 evaluable tumors/group) in 1) control; 2) MK-1775 (30 mg/kg, p.o., twice a week) for 4 weeks; 3) gemcitabine (100 mg/kg, i.p., twice a week) for 4 weeks; 4) gemcitabine followed 24 h later by MK-1775 in the above dose and frequency. Tumor growth rate/regressions were calculated on day 28. Target modulation by MK-1775 was assessed by western blot and IHC. Results: MK-1775 treatment leads to the inhibition of Wee1 kinase and its substrate phospho-cdc2. Combination of gemcitabine with MK-1775 abrogated the G2 arrest to promote the mitotic entry and facilitate tumor cell death. Although, MK-1775 monotherapy resulted in >50% inhibition of tumor growth in three of ten xenografts compared to control tumors, it did not result in tumor regressions. However, combination of gemcitabine with MK-1775 produced robust anti-tumor activity and remarkable increase in tumor regression (3.39 fold) in p53 mutated tumors compared to gemcitabine treatment. Tumor re-growth curves after the treatment suggest that the effect of the combination therapy is longer-lasting than that of gemcitabine. None of the agents or combination therapy produced tumor regressions in p53 wild type cases. Conclusions: Our results provide compelling evidence that MK-1775 synergizes with gemcitabine to achieve tumor regressions, selectively in p53 mutated pancreatic tumor xenografts and this combination merits clinical testing. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A251.

Discovery of gene expression-based pharmacodynamic biomarker for a p53 context-specific anti-tumor drug Wee1 inhibitor

BackgroundWee1 is a tyrosine kinase regulating S-G2 cell cycle transition through the inactivating phosphorylation of CDC2. The inhibition of Wee1 kinase by a selective small molecule inhibitor significantly enhances the anti-tumor efficacy of DNA damaging agents, specifically in p53 negative tumors by abrogating S-G2 checkpoints, while normal cells with wild-type p53 are not severely damaged due to the intact function of the G1 checkpoint mediated by p53. Since the measurement of mRNA expression requires a very small amount of biopsy tissue and is highly quantitative, the development of a pharmacodynamic (PD) biomarker leveraging mRNA expression is eagerly anticipated in order to estimate target engagement of anti-cancer agents.ResultsIn order to find the Wee1 inhibition signature, mRNA expression profiling was first performed in both p53 positive and negative cancer cell lines treated with gemcitabine and a Wee1 inhibitor, MK-1775. We next carried out mRNA expression profiling of skin samples derived from xenograft models treated with the Wee1 inhibitor to identify a Wee1 inhibitor-regulatory gene set. Then, the genes that were commonly modulated in both cancer cell lines and rat skin samples were extracted as a Wee1 inhibition signature that could potentially be used as a PD biomarker independent of p53 status. The expression of the Wee1 inhibition signature was found to be regulated in a dose-dependent manner by the Wee1 inhibitor, and was significantly correlated with the inhibition level of a direct substrate, phosphorylated-CDC2. Individual genes in this Wee1 inhibition signature are known to regulate S-G2 cell cycle progression or checkpoints, which is consistent with the mode-of-action of the Wee1 inhibitor.ConclusionWe report here the identification of an mRNA gene signature that was specifically changed by gemcitabine and Wee1 inhibitor combination treatment by molecular profiling. Given the common regulation of expression in both xenograft tumors and animal skin samples, the data suggest that the Wee1 inhibition gene signature might be utilized as a quantitative PD biomarker in both tumors and surrogate tissues, such as skin and hair follicles, in human clinical trials.

Cyclin B1 is an efficacy-predicting biomarker for Chk1 inhibitors

Chk1 is the major mediator of cell-cycle checkpoints in response to various forms of genotoxic stress. Although it was previously speculated that checkpoint abrogation due to Chk1 inhibition may potentiate the efficacy of DNA-damaging agents through induction of mitotic catastrophe, there has not been direct evidence proving this process. Here, through both molecular marker and morphological analysis, we directly demonstrate that specific downregulation of Chk1 expression by Chk1 siRNA potentiates the cytotoxicities of topoisomerase inhibitors through the induction of premature chromosomal condensation and mitotic catastrophe. More importantly, we discovered that the cellular cyclin B1 level is the major determinant of the potentiation. We show that downregulation of cyclin B1 leads to impairment of the induction of mitotic catastrophe and correspondingly a reduction of the potentiation ability of either Chk1 siRNA or a small molecule Chk1 inhibitor. More significantly, we have extended the study by examining a panel of 10 cancer cell-lines with different tissue origins for their endogenous levels of cyclin B1 and the ability of a Chk1 inhibitor to sensitize the cells to DNA-damaging agents. The cellular levels of cyclin B1 positively correlate with the degrees of potentiation achieved. Of additional interest, we observed that the various colon cancer cell lines in general appear to express higher levels of cyclin B1 and also display higher sensitivity to Chk1 inhibitors, implying that Chk1 inhibitor may be more efficacious in treating colon cancers. In summary, we propose that cyclin B1 is a biomarker predictive of the efficacy of Chk1 inhibitors across different types of cancers. Unlike previously established efficacy-predictive biomarkers that are usually the direct targets of the therapeutic agents, cyclin B1 represents a non-drug-target biomarker that is based on the mechanism of action of the target inhibitor. This finding may be potentially very useful for the stratification of patients for Chk1 inhibitor clinical trials and hence, maximize its chance of success.

Chk1 Inhibitors for Novel Cancer Treatment

Chemo- and radiotherapies that target DNA are the mainstay of cancer treatment. In response to DNA damage, cells are arrested in multiple checkpoints in the cell cycle to allow the damaged DNA to be repaired before progressing into mitosis. Normal cells are arrested in the G1 phase mediated by the p53 tumor suppressor, and p53-deficient cancer cells are arrested in the S or G2 phase. Checkpoint kinase 1 (Chk 1) is a serine / threonine protein kinase and a key mediator in the DNA damage-induced checkpoint network. When the G2 or S checkpoint is abrogated by the inhibition of Chk1, p53-deficient cancer cells undergo mitotic catastrophe and eventually apoptosis, whereas normal cells are still arrested in the G1 phase. Thus, Chk1 inhibitors can preferentially potentiate the efficacy of DNA damaging agents in cancer cells, and Chk1 is an attractive therapeutic target for cancer treatment, especially since approximately 50% of all human cancers are p53-deficient. This review discusses the rationale of Chk1 as an anticancer target, the structural basis for designing Chk1 inhibitors, and recently disclosed Chk1 inhibitors.

Evaluation of glucocorticoid sensitivity in 697 pre-B acute lymphoblastic leukemia cells after overexpression or silencing of MAP kinase phosphatase-1

To determine the effect of modulating MAP kinase phosphatase-1 (MKP-1) expression levels on cell death induced by glucocorticoid (GC) or hydroxyurea (HU) treatment in the human pre-B acute lymphoblastic leukemia cell line 697. Stable MKP-1 overexpressing transformants of the 697 pre-B acute lymphoblastic leukemia cell line were created and tested for sensitivity to the GC triamcinolone acetonide (TA) and HU, and compared to a control 697 cell line containing normal MKP-1 expression levels. Small interfering RNAs (siRNAs) were designed to inhibit MKP-1 expression and evaluated for their effect on GC-mediated cell death. MKP-1 overexpression caused a phenotype of partial resistance to HU-induced apoptosis but not to GC-induced apoptosis. Electroporation of siRNAs effectively silenced MKP-1 expression, and increased sensitivity to TA by 9.6+/-1.9%. Because MKP-1 protects certain tumor cells from chemotherapy-induced apoptosis, its inhibition is being considered as a possible strategy for combination cancer therapy. However, this study suggests that while MKP-1 inhibition may improve the efficacy of DNA damaging agents, it may have only limited utility in combination with glucocorticoids.