Emily Hanan

Discovery of Potent and Selective Pyrazolopyrimidine Janus Kinase 2 Inhibitors

The discovery of somatic Jak2 mutations in patients with chronic myeloproliferative neoplasms has led to significant interest in discovering selective Jak2 inhibitors for use in treating these disorders. A high-throughput screening effort identified the pyrazolo[1,5-a]pyrimidine scaffold as a potent inhibitor of Jak2. Optimization of lead compounds 7a-b and 8 in this chemical series for activity against Jak2, selectivity against other Jak family kinases, and good in vivo pharmacokinetic properties led to the discovery of 7j. In a SET2 xenograft model that is dependent on Jak2 for growth, 7j demonstrated a time-dependent knock-down of pSTAT5, a downstream target of Jak2.

Discovery and Optimization of C-2 Methyl Imidazopyrrolopyridines as Potent and Orally Bioavailable JAK1 Inhibitors with Selectivity over JAK2.

Herein we report the discovery of the C-2 methyl substituted imidazopyrrolopyridine series and its optimization to provide potent and orally bioavailable JAK1 inhibitors with selectivity over JAK2. The C-2 methyl substituted inhibitor 4 exhibited not only improved JAK1 potency relative to unsubstituted compound 3 but also notable JAK1 vs JAK2 selectivity (20-fold and >33-fold in biochemical and cell-based assays, respectively). Features of the X-ray structures of 4 in complex with both JAK1 and JAK2 are delineated. Efforts to improve the in vitro and in vivo ADME properties of 4 while maintaining JAK1 selectivity are described, culminating in the discovery of a highly optimized and balanced inhibitor (20). Details of the biological characterization of 20 are disclosed including JAK1 vs JAK2 selectivity levels, preclinical in vivo PK profiles, performance in an in vivo JAK1-mediated PK/PD model, and attributes of an X-ray structure in complex with JAK1.

4-Aminoindazolyl-dihydrofuro[3,4-d]pyrimidines as non-covalent inhibitors of mutant epidermal growth factor receptor tyrosine kinase.

The treatment of epidermal growth factor receptor (EGFR)-driven non-small cell lung cancers with the T790M resistance mutation remains a significant unmet medical need. We report the identification of 4-aminoindazolyl-dihydrofuro[3,4-d]pyrimidines as non-covalent inhibitors of EGFR, with excellent activity against the T790M resistance double mutants and initial single activating mutants. Using an optimization strategy focused on structure-based design and improving PK properties through metabolite identification, we obtained advanced leads with high oral exposure.

Abstract A20: Development and validation of a novel acute myeloid leukemia xenograft model that is dependent on the JAK2V617F mutation for growth in vivo

Background: The Janus‐associated kinase (JAK) proteins are a family of non‐receptor tyrosine kinases (JAK1, JAK2, JAK3, and TYK2) that play an important role in cellular survival, proliferation, and differentiation. A gain of function mutation (V617F) in the pseudo‐kinase domain of JAK2 is detected at high frequency in Philadelphia‐negative myeloproliferative disorders such as polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis. To further elucidate the role of JAK2V617F on cell growth in vivo we established and validated a novel xenograft model using the acute myeloid leukemia cell line, SET2, which is heterozygous for the mutation. Materials and Methods: SET2 cells containing stable doxycycline‐inducible JAK2V617F shRNA clones were generated by lentiviral transduction. Cell proliferation and viability were assessed by incorporation of 3H‐Thymidine or by ATP quantitation. Cell‐cycle and apoptosis markers were evaluated by Western blotting using antibodies against cyclin D1, PIM1, cleaved caspase 3 and 7 and PARP. Phospho‐STAT5 and total STAT5 were measured using both western blotting and ELISA. A SET2 shRNA clone was selected for in vivo growth in SCID beige female mice and treated with doxycycline or a JAK2 inhibitor (JAK2i). Plasma and tumor drug levels were measured by LC/MS. Results: Genetic knockdown using a doxycycline‐inducible JAK2V617F shRNA confirmed SET2 cells are dependent on this gain of function mutation for growth based on inhibition of cell proliferation in the presence of doxycycline. The latter was corroborated with a selective JAK2i (Ki= 4nM) that potently inhibited SET2 cell viability (EC50 = 355nM). The JAK2i rapidly suppressed phosphorylation of STAT5 (EC50 = 313nM) and induced cycle arrest at the G0–G1 phase based on downregulation of cyclin D1 expression. The JAK2i also effectively inhibited expression of PIM‐1 kinase, a STAT5 target gene, and activated cleavage of procaspase 3 and 7 and DNA‐repair enzyme PARP to induce apoptosis in a time‐ and dose‐dependent manner. A SET2 xenograft model was established by in vivo selection of a variant of the doxycycline‐ JAK2V617F shRNA parental cell line. Its dependence on JAK2V617F was confirmed by treatment with doxcycline and oral administration of a JAK2i, which resulted in significant tumor growth inhibition in vivo. Investigation of the PK/PD relationship underlying the activity of JAK2i provided direct evidence of phospho‐STAT5 suppression and induction of apoptosis for its antitumor effect in vivo. Plasma drug levels correlated with the duration and magnitude of suppression. Conclusion: A JAK2V617F dependent SET2 xenograft model has been established and validated by shRNA gene knockdown and pharmacologically with a JAK2i in vivo. JAK2V617F inhibition results in suppression of STAT5 phosphorylation, cell‐cycle arrest and induction of apoptosis in vitro and in vivo. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A20.

Abstract 156: Preclinical characterization of GDC-0077, a specific PI3K alpha inhibitor in early clinical development

The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/ mammalian target of rapamycin (mTOR) signaling pathway is a major regulator of tumor cell growth, proliferation and survival. Dysregulation of the PI3K/Akt/mTOR signaling pathway through multiple mechanisms has been described in solid tumor malignancies, including activating and transforming “hotspot” mutations as well as amplification of PIK3CA that encodes the p110 alpha subunit of PI3K. Hotspot mutations of PIK3CA mutation are frequently observed in breast cancer with a prevalence of approximately 30%. GDC-0077 is a potent inhibitor of PI3K alpha (IC50 = 0.038 + 0.003 nM) and exerts its activity by binding to the ATP binding site of PI3K, thereby inhibiting the phosphorylation of PIP2 to PIP3. Biochemically, GDC-0077 is more than 300-fold selective over other Class I PI3K isoforms such as beta, delta, and gamma and more than 2000 fold more selective over PI3K class II and III family members. Importantly, GDC-0077 is more selective for mutant versus wild-type PI3K alpha in cell based assays. Compared to the PI3K inhibitor, taselisib, the improved biochemical selectivity of GDC-0077 against PI3K delta is demonstrated in human CD69+ B-cells, which are primarily dependent on PI3K delta for proliferation and survival and were more sensitive to taselisib than GDC-0077. Mechanism of action studies indicate that GDC-0077 induces depletion of mutant PI3K alpha protein resulting in reduction of PI3K pathway biomarkers such as pAkt and pPRAS40, inhibition of cell proliferation and increased apoptosis in human PIK3CA mutant breast cancer cell lines to a greater extent when compared to PIK3CA wild-type cells. In vivo, daily oral treatment with GDC-0077 in cell-culture-derived and patient derived PIK3CA mutant breast cancer xenograft models, resulted in tumor regressions, induction of apoptosis and a reduction of pAkt, pPRAS40, and pS6RP in a dose-dependent fashion. In vivo efficacy in a PIK3CA-mutant human breast cancer xenograft model was also improved when GDC-0077 was combined with standard-of-care therapies for hormone-receptor positive (HR+) breast cancer such as anti-estrogens (fulvestrant) or CDK4/6 inhibitor (palbociclib). Collectively, the preclinical data provide rationale for evaluating GDC-0077, a PI3K alpha mutant selective inhibitor, as a single agent and in combination with standard-of-care endocrine and targeted therapies that may provide additional benefit to patients that harbor PIK3CA mutations. Citation Format: Kyle Edgar, Emily Hanan, Steven Staben, Stephen Schmidt, Rebecca Hong, Kyung Song, Amy Young, Patricia Hamilton, Alfonso Arrazate, Cecile de la Cruz, Marcia Belvin, Michelle Nannini, Lori S. Friedman, Deepak Sampath. Preclinical characterization of GDC-0077, a specific PI3K alpha inhibitor in early clinical development [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 156. doi:10.1158/1538-7445.AM2017-156

Abstract 1735: Discovery of novel and selective reversible inhibitors of EGFR containing the T790M drug resistance mutation with activity in vitro and in vivo

Activating mutations in the epidermal growth factor receptor (EGFR) characterize a subset of non-small cell lung cancers (NSCLC) with exquisite sensitivity to tyrosine kinase inhibitors like erlotinib or gefitinib. However, after an initial dramatic response, patients harboring these mutations progress within 8-14 months on therapy. In approximately 60% of patients, the resistance to TKIs is associated with the acquisition of a secondary mutation within the EGFR kinase domain. The mutation in the kinase domain leads to the substitution of a bulky methionine for threonine at position 790 (T790M) also referred to as the gatekeeper residue. Using structure based-design we identified and optimized a series of non-covalent, and highly selective pyridinyl-aminopyrimidine inhibitors of EGFR containing the T790M drug resistant mutation that spared the wild-type EGFR enzyme. These pyridinyl-aminopyrimidine compounds potently inhibited phosphorylation of T790M containing EGFR in enzymatic assays and cell based assays. Strong inhibition of enzyme activity resulted in anti-proliferative activity in H1975 and PC9 erlotinib resistant cells but treatment with these molecules did not affect cell lines containing wild-type EGFR. Moreover, oral dosing of mice bearing either H1975 or PC9_erlotinib resistant xenograft tumors resulted in robust inhibition of tumor growth that was accompanied by profound and dose-dependent inhibition of EGFR activity as well as downstream signaling molecules such as ERK1/2 and AKT. These results suggest that these pyridinyl-aminopyrimidine inhibitors have the potential to benefit NSCLC patients who have developed resistance to first-generation EGFR inhibitors due to the acquisition of the T790M mutation, without the skin rash and gastrointestinal toxicity associated with wild-type EGFR inhibition. *EJH and GS contributed equally to this work. Citation Format: Gabriele Schaefer, Emily J. Hanan, Emily Chan, Lily Shao, Yuan Chen, Jamie Knight, Robert L. Yauch, Stephen Schmidt, Steven Sideris, Shiva Malek, Timothy P. Heffron. Discovery of novel and selective reversible inhibitors of EGFR containing the T790M drug resistance mutation with activity in vitro and 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 1735. doi:10.1158/1538-7445.AM2014-1735