Valeria R. Fantin

PF-06463922, an ALK/ROS1 Inhibitor, Overcomes Resistance to First and Second Generation ALK Inhibitors in Preclinical Models

We report the preclinical evaluation of PF-06463922, a potent and brain-penetrant ALK/ROS1 inhibitor. Compared with other clinically available ALK inhibitors, PF-06463922 displayed superior potency against all known clinically acquired ALK mutations, including the highly resistant G1202R mutant. Furthermore, PF-06463922 treatment led to regression of EML4-ALK-driven brain metastases, leading to prolonged mouse survival, in a superior manner. Finally, PF-06463922 demonstrated high selectivity and safety margins in a variety of preclinical studies. These results suggest that PF-06463922 will be highly effective for the treatment of patients with ALK-driven lung cancers, including those who relapsed on clinically available ALK inhibitors because of secondary ALK kinase domain mutations and/or brain metastases.

PF-06463922 is a potent and selective next-generation ROS1/ALK inhibitor capable of blocking crizotinib-resistant ROS1 mutations

Significance Overcoming resistance to targeted kinase inhibitors is a major clinical challenge in oncology. Development of crizotinib resistance through the emergence of a secondary ROS1 mutation, ROS1G2032R, was observed in patients with ROS1 fusion-positive lung cancer. In addition, a novel ROS1 fusion recently has been identified in glioblastoma. A new agent with robust activity against the ROS1G2032R mutation and with CNS activity is needed to address these unmet medical needs. Here we report the identification of PF-06463922, a ROS1/anaplastic lymphoma kinase (ALK) inhibitor, with exquisite potency against ROS1 fusion kinases, capable of inhibiting the ROS1G2032R mutation and FIG-ROS1–driven glioblastoma tumor growth in preclinical models. PF-06463922 demonstrated excellent therapeutic potential against ROS1 fusion-driven cancers, and it currently is undergoing phase I/II clinical trial investigation. Oncogenic c-ros oncogene1 (ROS1) fusion kinases have been identified in a variety of human cancers and are attractive targets for cancer therapy. The MET/ALK/ROS1 inhibitor crizotinib (Xalkori, PF-02341066) has demonstrated promising clinical activity in ROS1 fusion-positive non-small cell lung cancer. However, emerging clinical evidence has shown that patients can develop resistance by acquiring secondary point mutations in ROS1 kinase. In this study we characterized the ROS1 activity of PF-06463922, a novel, orally available, CNS-penetrant, ATP-competitive small-molecule inhibitor of ALK/ROS1. In vitro, PF-06463922 exhibited subnanomolar cellular potency against oncogenic ROS1 fusions and inhibited the crizotinib-refractory ROS1G2032R mutation and the ROS1G2026M gatekeeper mutation. Compared with crizotinib and the second-generation ALK/ROS1 inhibitors ceritinib and alectinib, PF-06463922 showed significantly improved inhibitory activity against ROS1 kinase. A crystal structure of the PF-06463922-ROS1 kinase complex revealed favorable interactions contributing to the high-affinity binding. In vivo, PF-06463922 showed marked antitumor activity in tumor models expressing FIG-ROS1, CD74-ROS1, and the CD74-ROS1G2032R mutation. Furthermore, PF-06463922 demonstrated antitumor activity in a genetically engineered mouse model of FIG-ROS1 glioblastoma. Taken together, our results indicate that PF-06463922 has potential for treating ROS1 fusion-positive cancers, including those requiring agents with CNS-penetrating properties, as well as for overcoming crizotinib resistance driven by ROS1 mutation.

Mechanisms of Resistance to Histone Deacetylase Inhibitors and Their Therapeutic Implications

Histone deacetylase inhibitors (HDI) are a promising new approach to the treatment of cancer. HDIs have been shown to induce differentiation, cell cycle arrest, and apoptosis in a variety of transformed cell lines; inhibit tumor growth in animal models; and show antitumor activity in clinical trials. Vorinostat, which has shown clinical responses in ∼30% of patients with advanced cutaneous T-cell lymphoma, is the first HDI approved for the treatment of cancer, and it is currently being evaluated in other indications. A better understanding of the molecular determinants of resistance to HDIs may provide the basis for therapeutic combinations with improved clinical efficacy. Poor response to treatment could be linked to systemic factors like pharmacokinetics or to tumor-specific factors both at the level of the malignant cells (tumor intrinsic) or the tumor microenvironment. This review focuses on the tumor intrinsic mechanisms of drug resistance (excluding mechanism of acquired resistance due to chronic exposure). In particular, attention is given to selected mechanisms that are relevant across chemical classes of HDIs and that can aid in the design of rational combination strategies.

Macroautophagy is dispensable for growth of KRAS mutant tumors and chloroquine efficacy

Significance Kirsten rat sarcoma (KRAS) mutant tumors are believed to depend on autophagy for growth and survival. This study details the unexpected finding that autophagy-related 7, an enzyme essential for macroautophagy, can be deleted in several KRAS-driven cancer lines without affecting growth in vitro or in vivo. These data indicate that KRAS mutation status does not predict cell-autonomous addiction to autophagy. Furthermore, this report addresses a long-standing question regarding the mechanism of chloroquine, a lysosomotropic agent often used to interrogate effects of autophagy inhibition. Although chloroquine is antiproliferative and synergizes with targeted anticancer drugs, these effects are independent of macroautophagy. Future studies are needed to identify appropriate genetic stratification parameters to predict efficacy toward chloroquine and to characterize such agents further as anticancer combination partners. Macroautophagy is a key stress-response pathway that can suppress or promote tumorigenesis depending on the cellular context. Notably, Kirsten rat sarcoma (KRAS)-driven tumors have been reported to rely on macroautophagy for growth and survival, suggesting a potential therapeutic approach of using autophagy inhibitors based on genetic stratification. In this study, we evaluated whether KRAS mutation status can predict the efficacy to macroautophagy inhibition. By profiling 47 cell lines with pharmacological and genetic loss-of-function tools, we were unable to confirm that KRAS-driven tumor lines require macroautophagy for growth. Deletion of autophagy-related 7 (ATG7) by genome editing completely blocked macroautophagy in several tumor lines with oncogenic mutations in KRAS but did not inhibit cell proliferation in vitro or tumorigenesis in vivo. Furthermore, ATG7 knockout did not sensitize cells to irradiation or to several anticancer agents tested. Interestingly, ATG7-deficient and -proficient cells were equally sensitive to the antiproliferative effect of chloroquine, a lysosomotropic agent often used as a pharmacological tool to evaluate the response to macroautophagy inhibition. Moreover, both cell types manifested synergistic growth inhibition when treated with chloroquine plus the tyrosine kinase inhibitors erlotinib or sunitinib, suggesting that the antiproliferative effects of chloroquine are independent of its suppressive actions on autophagy.

Glutamine deprivation stimulates mTOR-JNK-dependent chemokine secretion

The non-essential amino acid, glutamine, exerts pleiotropic effects on cell metabolism, signalling and stress resistance. Here we demonstrate that short-term glutamine restriction triggers an endoplasmic reticulum (ER) stress response that leads to production of the pro-inflammatory chemokine, interleukin-8 (IL-8). Glutamine deprivation-induced ER stress triggers colocalization of autophagosomes, lysosomes and the Golgi into a subcellular structure whose integrity is essential for IL-8 secretion. The stimulatory effect of glutamine restriction on IL-8 production is attributable to depletion of tricarboxylic acid cycle intermediates. The protein kinase, mTOR, is also colocalized with the lysosomal membrane clusters induced by glutamine deprivation, and inhibition of mTORC1 activity abolishes both endomembrane reorganization and IL-8 secretion. Activated mTORC1 elicits IL8 gene expression via the activation of an IRE1-JNK signalling cascade. Treatment of cells with a glutaminase inhibitor phenocopies glutamine restriction, suggesting that these results will be relevant to the clinical development of glutamine metabolism inhibitors as anticancer agents.

Reciprocal regulation of amino acid import and epigenetic state through Lat1 and EZH2

Lat1 (SLC7A5) is an amino acid transporter often required for tumor cell import of essential amino acids (AA) including Methionine (Met). Met is the obligate precursor of S‐adenosylmethionine (SAM), the methyl donor utilized by all methyltransferases including the polycomb repressor complex (PRC2)‐specific EZH2. Cell populations sorted for surface Lat1 exhibit activated EZH2, enrichment for Met‐cycle intermediates, and aggressive tumor growth in mice. In agreement, EZH2 and Lat1 expression are co‐regulated in models of cancer cell differentiation and co‐expression is observed at the invasive front of human lung tumors. EZH2 knockdown or small‐molecule inhibition leads to de‐repression of RXRα resulting in reduced Lat1 expression. Our results describe a Lat1‐EZH2 positive feedback loop illustrated by AA depletion or Lat1 knockdown resulting in SAM reduction and concomitant reduction in EZH2 activity. shRNA‐mediated knockdown of Lat1 results in tumor growth inhibition and points to Lat1 as a potential therapeutic target.

Design and Synthesis of Pyridone-Containing 3,4-Dihydroisoquinoline-1(2H)-ones as a Novel Class of Enhancer of Zeste Homolog 2 (EZH2) Inhibitors

A new enhancer of zeste homolog 2 (EZH2) inhibitor series comprising a substituted phenyl ring joined to a dimethylpyridone moiety via an amide linkage has been designed. A preferential amide torsion that improved the binding properties of the compounds was identified for this series via computational analysis. Cyclization of the amide linker resulted in a six-membered lactam analogue, compound 18. This transformation significantly improved the ligand efficiency/potency of the cyclized compound relative to its acyclic analogue. Additional optimization of the lactam-containing EZH2 inhibitors focused on lipophilic efficiency (LipE) improvement, which provided compound 31. Compound 31 displayed improved LipE and on-target potency in both biochemical and cellular readouts relative to compound 18. Inhibitor 31 also displayed robust in vivo antitumor growth activity and dose-dependent de-repression of EZH2 target genes.

Abstract A277: PF-06463922, a novel ROS1/ALK inhibitor, demonstrates sub-nanomolar potency against oncogenic ROS1 fusions and capable of blocking the resistant ROS1G2032R mutant in preclinical tumor models.

The oncogenic ROS1 gene fusion ( Fig-ROS1 ) was first identified in glioblastoma cells over two decades ago. Recently, ROS1 gene rearrangements were further discovered in a variety of human cancers, including lung adenocarcinoma, cholangiocarcinoma, ovarian cancer, gastric adenocarcinoma, colorectal cancer, inflammatory myofibroblastic tumor, angiosarcoma, and epithelioid hemangioendothelioma, providing additional evidence for ROS1 as an attractive cancer target. The 1st generation Met/ALK/ROS1 inhibitor XALKORI ® (crizotinib) has demonstrated promising clinical response in ROS1 fusion positive NSCLC. But similar to what was seen with acquired ALK secondary resistant mutations in XALKORI refractory patients, a ROS1 kinase domain mutant–ROS1G2032R has been identified in a ROS1 positive NSCLC patient who developed resistance to XALKORI. Therefore, there is an urgent need to develop agents that can overcome this type of resistance. PF-06463922 is a novel, orally available, ATP-competitive small molecule inhibitor of ROS1/ALK with exquisite potency against ROS1 kinase. PF-06463922 inhibited the catalytic activity of recombinant ROS1 with a mean Ki of < 0.005 nM, and inhibited ROS1 autophosphorylation at IC50 values ranging from 0.1 nM to 1 nM cross a panel of cell lines harboring oncogenic ROS1 fusion variants including CD74-ROS1, SLC34A2-ROS1 and Fig-ROS1. PF-06463922 also inhibited cell proliferation and induced cell apoptosis at sub- to low-nanomolar concentrations in the HCC78 human NSCLC cells harboring SLC34A2-ROS1 fusions and the BaF3-CD74-ROS1 cells expressing human CD74-ROS1. In the BaF3 cells engineered to express the XALKORI resistant CD74-ROS1G2032R mutant, PF-06463922 demonstrated nanomolar potency against either ROS1G2032R cellular activity or cell proliferation. In vivo, PF-06463922 demonstrated marked cytoreductive antitumor efficacy at low nanomolar concentration in the NIH3T3 xenograft models expressing human CD74-ROS1 and Fig-ROS1. The antitumor efficacy of PF-06463922 was dose dependent and strongly correlated to inhibition in ROS1 phosphorylation and the downstream signaling molecules pSHP1, pSHP2 and pErk1/2, as well as inhibition of the cell cycle protein Cyclin D1 in tumors. To our knowledge, PF-06463922 is the first reported ROS1 inhibitor that is capable of blocking the resistant ROS1G2032R mutant at predicted pharmacologically relevant concentrations. Our data indicate that PF-06463922 has great potential for treating ROS1 fusion positive cancers including those from patients who relapsed from XALKORI therapy due to acquired ROS1G2032Rmutation. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A277. Citation Format: Helen Y. Zou, Lars R. Engstrom, Qiuhua Li, Melissa West Lu, Ruth Wei Tang, Hui Wang, Konstantinos Tsaparikos, Sergei Timofeevski, Justine Lam, Shinji Yamazaki, Wenyue Hu, Hovhannes Gukasyan, Nathan Lee, Ted W. Johnson, Valeria Fantin, Tod Smeal. PF-06463922, a novel ROS1/ALK inhibitor, demonstrates sub-nanomolar potency against oncogenic ROS1 fusions and capable of blocking the resistant ROS1G2032R mutant in preclinical tumor models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A277.

Targeting S -adenosylmethionine biosynthesis with a novel allosteric inhibitor of Mat2A

S-Adenosyl-L-methionine (SAM) is an enzyme cofactor used in methyl transfer reactions and polyamine biosynthesis. The biosynthesis of SAM from ATP and L-methionine is performed by the methionine adenosyltransferase enzyme family (Mat; EC 2.5.1.6). Human methionine adenosyltransferase 2A (Mat2A), the extrahepatic isoform, is often deregulated in cancer. We identified a Mat2A inhibitor, PF-9366, that binds an allosteric site on Mat2A that overlaps with the binding site for the Mat2A regulator, Mat2B. Studies exploiting PF-9366 suggested a general mode of Mat2A allosteric regulation. Allosteric binding of PF-9366 or Mat2B altered the Mat2A active site, resulting in increased substrate affinity and decreased enzyme turnover. These data support a model whereby Mat2B functions as an inhibitor of Mat2A activity when methionine or SAM levels are high, yet functions as an activator of Mat2A when methionine or SAM levels are low. The ramification of Mat2A activity modulation in cancer cells is also described.

Targeting the CXCR4 pathway using a novel anti-CXCR4 IgG1 antibody (PF-06747143) in chronic lymphocytic leukemia

BackgroundThe CXCR4-CXCL12 axis plays an important role in the chronic lymphocytic leukemia (CLL)-microenvironment interaction. Overexpression of CXCR4 has been reported in different hematological malignancies including CLL. Binding of the pro-survival chemokine CXCL12 with its cognate receptor CXCR4 induces cell migration. CXCL12/CXCR4 signaling axis promotes cell survival and proliferation and may contribute to the tropism of leukemia cells towards lymphoid tissues and bone marrow. Therefore, we hypothesized that targeting CXCR4 with an IgG1 antibody, PF-06747143, may constitute an effective therapeutic approach for CLL.MethodsPatient-derived primary CLL-B cells were assessed for cytotoxicity in an in vitro model of CLL microenvironment. PF-06747143 was analyzed for cell death induction and for its potential to interfere with the chemokine CXCL12-induced mechanisms, including migration and F-actin polymerization. PF-06747143 in vivo efficacy was determined in a CLL murine xenograft tumor model.ResultsPF-06747143, a novel-humanized IgG1 CXCR4 antagonist antibody, induced cell death of patient-derived primary CLL-B cells, in presence or absence of stromal cells. Moreover, cell death induction by the antibody was independent of CLL high-risk prognostic markers. The cell death mechanism was dependent on CXCR4 expression, required antibody bivalency, involved reactive oxygen species production, and did not require caspase activation, all characteristics reminiscent of programmed cell death (PCD). PF-06747143 also induced potent B-CLL cytotoxicity via Fc-driven antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity activity (CDC). PF-06747143 had significant combinatorial effect with standard of care (SOC) agents in B-CLL treatment, including rituximab, fludarabine (F-ara-A), ibrutinib, and bendamustine. In a CLL xenograft model, PF-06747143 decreased tumor burden and improved survival as a monotherapy, and in combination with bendamustine.ConclusionsWe show evidence that PF-06747143 has biological activity in CLL primary cells, supporting a rationale for evaluation of PF-06747143 for the treatment of CLL patients.