Shannon Marie Karlicek

Small-molecule p21-activated kinase inhibitor PF-3758309 is a potent inhibitor of oncogenic signaling and tumor growth

Despite abundant evidence that aberrant Rho-family GTPase activation contributes to most steps of cancer initiation and progression, there is a dearth of inhibitors of their effectors (e.g., p21-activated kinases). Through high-throughput screening and structure-based design, we identify PF-3758309, a potent (Kd = 2.7 nM), ATP-competitive, pyrrolopyrazole inhibitor of PAK4. In cells, PF-3758309 inhibits phosphorylation of the PAK4 substrate GEF-H1 (IC50 = 1.3 nM) and anchorage-independent growth of a panel of tumor cell lines (IC50 = 4.7 ± 3 nM). The molecular underpinnings of PF-3758309 biological effects were characterized using an integration of traditional and emerging technologies. Crystallographic characterization of the PF-3758309/PAK4 complex defined determinants of potency and kinase selectivity. Global high-content cellular analysis confirms that PF-3758309 modulates known PAK4-dependent signaling nodes and identifies unexpected links to additional pathways (e.g., p53). In tumor models, PF-3758309 inhibits PAK4-dependent pathways in proteomic studies and regulates functional activities related to cell proliferation and survival. PF-3758309 blocks the growth of multiple human tumor xenografts, with a plasma EC50 value of 0.4 nM in the most sensitive model. This study defines PAK4-related pathways, provides additional support for PAK4 as a therapeutic target with a unique combination of functions (apoptotic, cytoskeletal, cell-cycle), and identifies a potent, orally available small-molecule PAK inhibitor with significant promise for the treatment of human cancers.

Enzymatic Characterization of c-Met Receptor Tyrosine Kinase Oncogenic Mutants and Kinetic Studies with Aminopyridine and Triazolopyrazine Inhibitors

The c-Met receptor tyrosine kinase (RTK) is a key regulator in cancer, in part, through oncogenic mutations. Eight clinically relevant mutants were characterized by biochemical, biophysical, and cellular methods. The c-Met catalytic domain was highly active in the unphosphorylated state (k(cat) = 1.0 s(-1)) and achieved 160-fold enhanced catalytic efficiency (k(cat)/K(m)) upon activation to 425000 s(-1) M(-1). c-Met mutants had 2-10-fold higher basal enzymatic activity (k(cat)) but achieved maximal activities similar to those of wild-type c-Met, except for Y1235D, which underwent a reduction in maximal activity. Small enhancements of basal activity were shown to have profound effects on the acquisition of full enzymatic activity achieved through accelerating rates of autophosphorylation. Biophysical analysis of c-Met mutants revealed minimal melting temperature differences indicating that the mutations did not alter protein stability. A model of RTK activation is proposed to describe how a RTK response may be matched to a biological context through enzymatic properties. Two c-Met clinical candidates from aminopyridine and triazolopyrazine chemical series (PF-02341066 and PF-04217903) were studied. Biochemically, each series produced molecules that are highly selective against a large panel of kinases, with PF-04217903 (>1000-fold selective relative to 208 kinases) being more selective than PF-02341066. Although these prototype inhibitors have similar potencies against wild-type c-Met (K(i) = 6-7 nM), significant differences in potency were observed for clinically relevant mutations evaluated in both biochemical and cellular contexts. In particular, PF-02341066 was 180-fold more active against the Y1230C mutant c-Met than PF-04217903. These highly optimized inhibitors indicate that for kinases susceptible to active site mutations, inhibitor design may need to balance overall kinase selectivity with the ability to inhibit multiple mutant forms of the kinase (penetrance).

Osteopontin induces growth of metastatic tumors in a preclinical model of non-small lung cancer

Osteopontin (OPN), also known as SPP1 (secreted phosphoprotein), is an integrin binding glyco-phosphoprotein produced by a variety of tissues. In cancer patients expression of OPN has been associated with poor prognosis in several tumor types including breast, lung, and colorectal cancers. Despite wide expression in tumor cells and stroma, there is limited evidence supporting role of OPN in tumor progression and metastasis. Using phage display technology we identified a high affinity a nti-O PN m onoclonal antibody (hereafter AOM1). The binding site for AOM1 was identified as SVVYGLRSKS sequence which is immediately adjacent to the RGD motif and also spans the thrombin cleavage site of the human OPN. AOM1 efficiently inhibited OPNa binding to recombinant integrin αvβ3 with an IC50 of 65 nM. Due to its unique binding site, AOM1 is capable of inhibiting OPN cleavage by thrombin which has been shown to produce an OPN fragment that is biologically more active than the full length OPN. Screening of human cell lines identified tumor cells with increased expression of OPN receptors (αvβ3 and CD44v6) such as mesothelioma, hepatocellular carcinoma, breast, and non-small cell lung adenocarcinoma (NSCLC). CD44v6 and αvβ3 were also found to be highly enriched in the monocyte, but not lymphocyte, subset of human peripheral blood mononuclear cells (hPBMCs). In vitro, OPNa induced migration of both tumor and hPBMCs in a transwell migration assay. AOM1 significantly blocked cell migration further validating its specificity for the ligand. OPN was found to be enriched in mouse plasma in a number of pre-clinical tumor model of non-small cell lung cancers. To assess the role of OPN in tumor growth and metastasis and to evaluate a potential therapeutic indication for AOM1, we employed a KrasG12D-LSLp53fl/fl subcutaneously implanted in vivo model of NSCLC which possesses a high capacity to metastasize into the lung. Our data indicated that treatment of tumor bearing mice with AOM1 as a single agent or in combination with Carboplatin significantly inhibited growth of large metastatic tumors in the lung further supporting a role for OPN in tumor metastasis and progression.

Abstract 4456: Biochemical and structural characteristics of crizotinib-resistant ALK mutants.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Anaplastic lymphoma kinase (ALK), when aberrantly regulated through gene translocations or mutations, has been implicated as an oncogenic driver in a variety of cancers. XALKORI (crizotinib) is an FDA-approved therapy for locally advanced or metastatic (ALK)-positive non-small cell lung cancer (NSCLC). Mutations of certain residues within the ALK kinase domain have been associated with crizotinib resistance. To understand the molecular basis of mutational resistance, we have kinetically and structurally characterized several clinical mutations of ALK kinase domain. In the basal-state (nonphosphorylated) proteins, mutations C1156Y, F1174L, L1152R and L1196M resulted in 14 to 52-fold increase in catalytic efficiency of phosphorylation of an activation loop peptide. Accordingly, these mutants were more rapidly autophosphorylated, compared to wild-type enzyme. In addition, F1174L, C1156Y and L1156Y had 2 to 6-fold higher catalytic efficiencies when fully activated by autophosphorylation. Conversely, catalytic efficiencies of nonphosphorylated and phosphorylated G1269A variant decreased by 1.2 and 1.6-fold, respectively. Inhibition of L1152R, L1196M and G1269A by crizotinib was reduced by 3-28-fold, compared to wild-type enzyme, from Ki determinations. From direct binding studies of the L1196M “gatekeeper” mutation, the loss of crizotinib binding occurred 6 and 13-fold for phosphorylated and nonphosphorylated proteins, respectively. By crystallographic studies apoenzyme and crizotinib complexes of ALK kinase domain displayed an inactive kinase conformation that is stabilized by an extended hydrophobic network of residues. F1174 and L1196 are part of this hydrophobic core, and mutations of these residues are predicted to destabilize the inactive ALK conformation. Taken together, these results suggest that most of the resistant mutations (e.g. L1196M, F1174L, C1156Y, L1152R) result in a more dynamic protein that increases substrate turnover. In addition, the reduction of crizotinib binding for the mutations in the vicinity of the inhibitor binding site (L1196M, G1269A) is likely a contributing factor for the resistance. This structural and kinetic analysis of mutational resistance may be useful for design of new inhibitors targeting multiple clinical mutations of ALK. Citation Format: Sergei Timofeevski, Wei Liu, Ya-Li Deng, Alexei Brooun, Simon Bergqvist, Shannon Karlicek, Brion Murray, Ben Bolanos, Michele McTigue. Biochemical and structural characteristics of crizotinib-resistant ALK mutants. [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 4456. doi:10.1158/1538-7445.AM2013-4456