Amy Jackson-Fisher

Mice lacking the epidermal growth factor receptor family member ErbB4 exhibit defects in cranial neural crest cell migration but die by embryonic day 11 because of defective heart development. To examine later phenotypes, we rescued the heart defects in ErbB4 mutant mice by expressing ErbB4 under a cardiac-specific myosin promoter. Rescued ErbB4 mutant mice reach adulthood and are fertile. However, during pregnancy, mammary lobuloalveoli fail to differentiate correctly and lactation is defective. Rescued mice also display aberrant cranial nerve architecture and increased numbers of large interneurons within the cerebellum.

First generation EGFR TKIs (gefitinib, erlotinib) provide significant clinical benefit for NSCLC cancer patients with oncogenic EGFR mutations. Ultimately, these patients' disease progresses, often driven by a second-site mutation in the EGFR kinase domain (T790M). Another liability of the first generation drugs is severe adverse events driven by inhibition of WT EGFR. As such, our goal was to develop a highly potent irreversible inhibitor with the largest selectivity ratio between the drug-resistant double mutants (L858R/T790M, Del/T790M) and WT EGFR. A unique approach to develop covalent inhibitors, optimization of reversible binding affinity, served as a cornerstone of this effort. PF-06459988 was discovered as a novel, third generation irreversible inhibitor, which demonstrates (i) high potency and specificity to the T790M-containing double mutant EGFRs, (ii) minimal intrinsic chemical reactivity of the electrophilic warhead, (iii) greatly reduced proteome reactivity relative to earlier irreversible EGFR inhibitors, and (iv) minimal activity against WT EGFR.

BACKGROUND: Dormant leukemia stem cells (LSC) promote therapeutic resistance and leukemic progression as a result of unbridled activation of stem cell gene expression programs. Thus, we hypothesized that 1) deregulation of the hedgehog (Hh) stem cell self-renewal and cell cycle regulatory pathway would promote dormant human LSC generation and 2) that PF-04449913, a clinical antagonist of the GLI2 transcriptional activator, smoothened (SMO), would enhance dormant human LSC eradication. METHODS: To test these postulates, whole transcriptome RNA sequencing (RNA-seq), microarray, qRT-PCR, stromal co-culture, confocal fluorescence microscopic, nanoproteomic, serial transplantation and cell cycle analyses were performed on FACS purified normal, chronic phase (CP) chronic myeloid leukemia (CML), blast crisis (BC) phase CML progenitors with or without PF-04449913 treatment. RESULTS: Notably, RNA-seq analyses revealed that Hh pathway and cell cycle regulatory gene overexpression correlated with leukemic progression. While lentivirally enforced GLI2 expression enhanced leukemic progenitor dormancy in stromal co-cultures, this was not observed with a mutant GLI2 lacking a transactivation domain, suggesting that GLI2 expression prevented cell cycle transit. Selective SMO inhibition with PF-04449913 in humanized stromal co-cultures and LSC xenografts reduced downstream GLI2 protein and cell cycle regulatory gene expression. Moreover, SMO inhibition enhanced cell cycle transit and sensitized BC LSC to tyrosine kinase inhibition in vivo at doses that spare normal HSC. CONCLUSION: In summary, while GLI2, forms part of a core HH pathway transcriptional regulatory network that promotes human myeloid leukemic progression and dormant LSC generation, selective inhibition with PF-04449913 reduces the dormant LSC burden thereby providing a strong rationale for clinical trials predicated on SMO inhibition in combination with TKIs or chemotherapeutic agents with the ultimate aim of obviating leukemic therapeutic resistance, persistence and progression.

The ERBB2/HER2/NEU receptor tyrosine kinase gene is amplified in up to 30% of human breast cancers. The frequent and specific selection of this receptor kinase gene for amplification in breast cancer implies that it has important normal functions in the mammary gland. To investigate the functions of ErbB2 during normal mouse mammary gland development, we transplanted mammary buds from genetically rescued ErbB2(-/-) embryos that express ErbB2 in the cardiac muscle. ErbB2(-/-) mammary buds transplanted to a wild-type mammary fat pad support outgrowth of an epithelial tree that advances only slowly through the mammary fat pad at puberty. This penetration defect is associated with structural defects in terminal end buds, characterized by a decrease in body cell number, an increased presence of cap-like cells in the prelumenal compartment, and the presence of large luminal spaces. Lobuloalveolar development was not affected in glands that developed from ErbB2(-/-) transplanted tissue. The results may have implications for the aggressive phenotypes associated with ERBB2-overexpressing mammary carcinomas.

Abstract Purpose: We investigated safety, tolerability, pharmacokinetics, and antitumor activity of the protein tyrosine kinase 7 (PTK7)-targeted, auristatin-based antibody–drug conjugate (ADC) PF-06647020/cofetuzumab pelidotin (NCT02222922). Patients and Methods: Patients received PF-06647020 intravenously every 3 weeks at 0.2–3.7 mg/kg or every 2 weeks at 2.1–3.2 mg/kg, in sequential dose escalation, following a modified toxicity probability interval method. In dose expansion, pretreated patients with advanced, platinum-resistant ovarian cancer, non–small cell lung cancer (NSCLC), or triple-negative breast cancer (TNBC) received PF-06647020 2.8 mg/kg every 3 weeks. Results: The most common, treatment-related adverse events for PF-06647020 administered every 3 weeks were nausea, alopecia, fatigue, headache, neutropenia, and vomiting (45%–25%); 25% of patients had grade ≥ 3 neutropenia. Two patients experienced dose-limiting toxicities (grade 3 headache and fatigue) at the highest every 3 weeks dose evaluated. The recommended phase II dose was 2.8 mg/kg every 3 weeks. The overall safety profile observed with PF-06647020 administered every 2 weeks was similar to that of the every 3 weeks regimen. Systemic exposure for the ADC and total antibody generally increased in a dose-proportional manner. Antitumor activity was observed in treated patients with overall objective response rates of 27% in ovarian cancer (n = 63), 19% in NSCLC (n = 31), and 21% in TNBC (n = 29). Responders tended to have moderate or high PTK7 tumor expression by IHC. Conclusions: This PTK7-targeted ADC demonstrated therapeutic activity in previously treated patients with ovarian cancer, NSCLC, and TNBC at a dose range of 2.1–3.2 mg/kg, supporting further clinical evaluation to refine dose, schedule, and predictive tissue biomarker testing in patients with advanced malignancies.