Stephane Thibault

Specific inhibition of Notch1 signaling enhances the antitumor efficacy of chemotherapy in triple negative breast cancer through reduction of cancer stem cells

Recent evidence suggests that Notch signaling may play a role in regulation of cancer stem cell (CSC) self-renewal and differentiation hence presenting a promising target for development of novel therapies for aggressive cancers such as triple negative breast cancer (TNBC). We generated Notch1 monoclonal antibodies (mAbs) that specifically bind to the negative regulatory region of human Notch1. Notch1 inhibition in TNBC Sum149 and patient derived xenograft (PDX) 144580 models led to significant TGI particularly in combination with docetaxel. More interestingly, Notch1 mAbs caused a reduction in mammosphere formation and CD44+/CD24-/lo cell population. It also resulted in decreased tumor incidence upon re-implantation and delay in tumor recurrence. Our data demonstrated a potent antitumor efficacy of Notch1 mAbs, with a remarkable activity against CSCs. These findings suggest that anti-Notch1 mAbs may provide novel therapies to improve the efficacy of conventional therapies by directly targeting the CSC niche. They may also delay tumor recurrence and hence have a major impact on cancer patient survival.

Mechanistic Investigation of Bone Marrow Suppression Associated with Palbociclib and its Differentiation from Cytotoxic Chemotherapies.

Purpose: Palbociclib (PD-0332991) is the first selective cyclin-dependent kinase (CDK) 4/6 inhibitor approved for metastatic breast cancer. Hematologic effects, especially neutropenia, are dose-limiting adverse events for palbociclib in humans. Experimental Design: Reversible hematologic effects and bone marrow hypocellularity have been identified in toxicology studies in rats and dogs after palbociclib treatment. To understand the mechanism by which the hematologic toxicity occurs, and to further differentiate it from the myelotoxicity caused by cytotoxic chemotherapeutic agents, an in vitro assay using human bone marrow mononuclear cells (hBMNC) was utilized. Results: This work demonstrated that palbociclib-induced bone marrow suppression occurred through cell-cycle arrest, with no apoptosis at clinically relevant concentrations, was not lineage-specific, and was reversible upon palbociclib withdrawal. In contrast, treatment with chemotherapeutic agents (paclitaxel and doxorubicin) resulted in DNA damage and apoptotic cell death in hBMNCs. In the presence or absence of the antiestrogen, palbociclib-treated hBMNCs did not become senescent and resumed proliferation following palbociclib withdrawal, consistent with pharmacologic quiescence. The breast cancer cells, MCF-7, conversely, became senescent following palbociclib or antiestrogen treatment with additive effects in combination and remained arrested in the presence of antiestrogen. Conclusions: Palbociclib causes reversible bone marrow suppression, clearly differentiating it from apoptotic cell death caused by cytotoxic chemotherapeutic agents. This study also distinguished the cell-cycle arresting action of palbociclib on normal bone marrow cells from the senescent effects observed in breast cancer cells. These results shed light on the mechanism and support risk management of palbociclib-induced bone marrow toxicity in the clinic. Clin Cancer Res; 22(8); 2000–8. ©2015 AACR.

Considerations for the nonclinical safety evaluation of antibody drug conjugates for oncology

Antibody drug conjugates (ADCs) include monoclonal antibodies that are linked to cytotoxic small molecules. A number of these agents are currently being developed as anti-cancer agents designed to improve the therapeutic index of the cytotoxin (i.e., cytotoxic small molecule or cytotoxic agent) by specifically delivering it to tumor cells. This paper presents primary considerations for the nonclinical safety evaluation of ADCs and includes strategies for the evaluation of the entire ADC or the various individual components (i.e., antibody, linker or the cytotoxin). Considerations are presented on how to design a nonclinical safety assessment program to identify the on- and off-target toxicities to enable first-in-human (FIH) studies. Specific discussions are also included that provide details as to the need and how to conduct the studies for evaluating ADCs in genetic toxicology, tissue cross-reactivity, safety pharmacology, carcinogenicity, developmental and reproductive toxicology, biotransformation, toxicokinetic monitoring, bioanalytical assays, immunogenicity testing, test article stability and the selection of the FIH dose. Given the complexity of these molecules and our evolving understanding of their properties, there is no single all-encompassing nonclinical strategy. Instead, each ADC should be evaluated on a case-by-case scientifically-based approach that is consistent with ICH and animal research guidelines.

Proliferative and Non-Proliferative Lesions of the Rat and Mouse Soft Tissue, Skeletal Muscle and Mesothelium

Abstract The INHAND Project (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) is a joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP), and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying lesions observed in the soft tissues including skeletal muscle as well as the mesothelium of rats and mice. The standardized nomenclature of lesions presented in this document is also available electronically on the Internet (http://www.goreni.org/). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous developmental and aging lesions as well as those induced by exposure to test materials. A widely accepted and utilized international harmonization of nomenclature for lesions in soft tissues, skeletal muscle and mesothelium in laboratory animals will decrease confusion among regulatory and scientific research organizations in different countries and provide a common language to increase and enrich international exchanges of information among toxicologists and pathologists. (DOI: 10.1293/tox.26.1S; J Toxicol Pathol 2013; 26: 1S–26S)

Evaluation of miR-216a and miR-217 as Potential Biomarkers of Acute Exocrine Pancreatic Toxicity in Rats:

Detecting and monitoring exocrine pancreatic damage during nonclinical and clinical testing is challenging because classical biomarkers amylase and lipase have limited sensitivity and specificity. Novel biomarkers for drug-induced pancreatic injury are needed to improve safety assessment and reduce late-stage attrition rates. In a series of studies, miR-216a and miR-217 were evaluated as potential biomarkers of acute exocrine pancreatic toxicity in rats. Our results revealed that miR-216a and miR-217 were almost exclusively expressed in rat pancreas and that circulating miR-216a and miR-217 were significantly increased in rats following administration of established exocrine pancreatic toxicants caerulein (CL) and 1-cyano-2-hydroxy-3-butene (CHB) as well as in rats administered a proprietary molecule known to primarily affect the exocrine pancreas. Conversely, neither microRNA was increased in rats administered a proprietary molecule known to cause a lesion at the pancreatic endocrine–exocrine interface (EEI) or in rats administered an established renal toxicant. Compared with amylase and lipase, increases in miR-216a and miR-217 were of greater magnitude, persisted longer, and/or correlated better with microscopic findings within the exocrine pancreas. Our findings demonstrate that in rats, miR-216a and miR-217 are sensitive and specific biomarkers of acute exocrine pancreatic toxicity that may add value to the measurement of classical pancreatic biomarkers.

Nonclinical assessments of the potential biosimilar PF-06439535 and bevacizumab

ABSTRACT Bevacizumab, a recombinant humanized monoclonal antibody targeting vascular endothelial growth factor (VEGF), is approved for treatment of metastatic colorectal cancer, nonsquamous non–small‐cell lung cancer, metastatic kidney cancer, and glioblastoma. To support clinical development of the potential bevacizumab biosimilar PF‐06439535, nonclinical studies evaluated structural, functional, toxicological, and toxicokinetic similarity to bevacizumab sourced from the European Union (bevacizumab‐EU) and United States (bevacizumab‐US). Peptide mapping demonstrated the amino acid sequence of PF‐06439535 was identical to bevacizumab‐EU and bevacizumab‐US. Biologic activity, measured via inhibition of VEGF‐induced cell proliferation in human umbilical vein endothelial cells and binding to VEGF isoforms, was similar across the three drugs. In vivo similarity was demonstrated in cynomolgus monkeys administered intravenous PF‐06439535 or bevacizumab‐EU (0 or 10 mg/kg/dose twice weekly for 1 month; total of nine doses). Systemic exposure appeared similar and test article–related effects were limited to physeal dysplasia of the distal femur. The potential for non–target‐mediated toxicity of PF‐06439535 was evaluated in rats administered intravenous PF‐06439535 (15 or 150 mg/kg/dose twice weekly for 15 days; total of five doses). Nonadverse higher liver weights and minimal sinusoidal cell hyperplasia were observed. Collectively, these studies demonstrated similarity of PF‐06439535 to bevacizumab, supporting entry into clinical development. HighlightsPF‐06439535 is a potential biosimilar to bevacizumab.The similarity of PF‐06439535 and bevacizumab was assessed by nonclinical analyses.PF‐06439535 and bevacizumab had similar primary structure, binding, and function.The toxicity of PF‐06439535 and bevacizumab in cynomolgus monkeys appeared similar.PF‐06439535 was well tolerated in rats.

Abstract 3095: The development of CPI as a novel, next-generation DNA-targeting payload for ADCs

DNA targeting drugs represent one of cornerstones of anti-cancer therapy for both hematologic and solid tumor indications. Low potency anti-DNA compounds (e.g. platins, anthracyclines) are included in many standard-of-care (SOC) regimens, however their modest activity and overall toxicity profiles limit their therapeutic potential. To increase the therapeutic window for DNA-damaging agents, high potency anti-DNA compounds with enhanced anti-tumor activity have been delivered to tumors as payloads of targeting modalities such as antibody-drug conjugates (ADCs). Herein, we describe the development of a novel DNA-damaging compound comprised of a dimeric structure of cyclopropylpyrrolo[e]indolones (CPIs) that was designed to alkylate DNA and generate toxic interstrand crosslinks (ICLs). In response to the CPI-induced formation of ICLs, CPI treatment of cells primarily activates the Fancomia anemia DNA damage response pathway, whereas other successful DNA-damaging ADC payloads such as calicheamicin activate double-strand break response pathways. CPI shows ~860-fold greater potency than calicheamicin in a panel of cell lines derived from a broad spectrum of tumor indications. Importantly, this new CPI payload retains potent activity in calicheamicin- and SOC-resistant tumor models (including overcoming overexpression of drug efflux pumps). When evaluated as payloads on anti-CD33 targeting ADCs, the CPI conjugate showed dramatically improved efficacy over the corresponding calicheamicin conjugates in MDR+ tumor models. As a site-specific conjugate, the CPI ADC shows enhanced in vivo stability and possesses a wider therapeutic window than the corresponding conventional calicheamicin conjugate and other leading DNA-damaging conjugates on the CD33 platform. Citation Format: Jennifer Kahler, Maureen Dougher, Jane Xu, Matthew Doroski, Andreas Maderna, Russell Dushin, Stephane Thibault, Mauricio Leal, Madan Katragadda, Christopher J. O9Donnell, Matthew Sung, Puja Sapra. The development of CPI as a novel, next-generation DNA-targeting payload for ADCs [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 3095. doi:10.1158/1538-7445.AM2017-3095

CDK4/6 Inhibition on Glucose and Pancreatic Beta Cell Homeostasis in Young and Aged Rats

Genetic deletion of cyclin-dependent kinase 4 (Cdk4) is associated with pancreatic beta cell loss and glucose dysregulation in rodents. Palbociclib, one of the first selective CDK4/6 inhibitors approved for the treatment of advanced breast cancer, is currently being investigated as an adjuvant treatment in patients with early-stage breast cancer and in a variety of cancers covering a wide-range of patient populations. Hence, longer chronic toxicity studies were necessary to further examine its safety profile. The effects of different doses and duration of palbociclib administration on glucose and beta cell homeostasis in young (two months) versus aged (12 months) rats was compared. Glucose dysregulation, due to pancreatic beta cell degeneration, was observed in young rats administered the highest dose of palbociclib for 6 months. Abnormal pancreatic islet histology and activation of the endoplasmic reticulum stress response in beta cells were detected after shorter administration with high-dose palbociclib in young rats. To test the hypothesis that palbociclib-associated inhibition of beta cell proliferation will more profoundly affect younger animals that have not achieved replicative quiescence, we administered high-dose palbociclib to aged rats for 6 months. In contrast to the young rats, despite equivalent exposures to palbociclib, no evidence of impaired glucose tolerance, hypoinsulinemia, beta cell vacuolization, or beta cell loss was seen in aged rats. Palbociclib administration induces beta cell failure in young but not aged rats. Implications: Although adult humans receiving palbociclib have not displayed detectable adverse effects on glucose metabolism, the risk of beta cell failure in children remains unexplored. Mol Cancer Res; 15(11); 1531–41. ©2017 AACR.

Abstract 3485: Inhibition of Notch signaling enhances the antitumor efficacy of chemotherapy in triple negative breast cancer through reduction of cancer stem cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Triple negative breast cancer (TNBC) is characterized by lack of estrogen receptor (ER), progesterone receptor (PR) or Her2/neu. It is known to be particularly aggressive and refractory to current chemotherapies. Tumor-initiating cells (TICs) have been postulated as responsible for treatment failure for this type of cancer. Recent evidence suggests that Notch signaling may play a role in regulation of TIC self-renewal and differentiation hence present a promising target for development of novel therapies for TNBC. We previously discovered a mouse monoclonal Ab (mu-hN1) that produces robust efficacy in both T-cell acute lymphoblastic leukemia and TNBC by specifically blocking human Notch1 signaling in tumor cells. We subsequently produced a humanized Notch1 mAb (hu-hN1) that retained similar characteristics and activities compared to the mouse prototype mu-hN1. To gain insights into the mechanisms of Notch1 blockade on TNBC tumor grow inhibition (TGI), we evaluated the effect of hu-hN1 on apoptosis, proliferation and TIC self-renewal in Notch-driven Sum149 and a patient derived xenograft (PDX) TNBC models. We showed that hu-hN1 inhibited Notch signaling by reduction of NICD (Notch intracellular domain) and target genes Hes-1 and cMyc in Sum149 tumors. Inhibition of Notch signaling led to significant TGI either as a single agent or in combination with Docetaxel. TGI of hu-hN1 appeared to be in part due to induction of apoptosis in the tumor cells. More interestingly, treatment with this Ab caused reduction in mammosphere formation and CD44+/CD133+/ESA+ cell population. It also resulted in decrease in tumor incidence and delay in tumor recurrence, suggesting an anti-TIC activity of the agent. Further investigation in a PDX TNBC model confirmed the robust tumor efficacy and anti-TIC effect of hu-hN1. Taken together, our findings suggest that anti-Notch1 mAbs may provide novel therapies to improve the efficacy of conventional treatments by directly targeting the TIC niche. They may also delay tumor recurrence and hence have a major impact on cancer patient survival. 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 3485. doi:1538-7445.AM2012-3485