Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are ...

University of Colorado Anschutz Medical Campus

Skeletal disorders and neural tube closure defects represent clinically significant human malformations. The signaling networks regulating normal skeletal patterning and neurulation are largely unknown. Targeted mutation of the active site lysine of MEK kinase 4 (MEKK4) produces a kinase-inactive MEKK4 protein (MEKK4(K1361R)). Embryos homozygous for this mutation die at birth as a result of skeletal malformations and neural tube defects. Hindbrains of exencephalic MEKK4(K1361R) embryos show a striking increase in neuroepithelial cell apoptosis and a dramatic loss of phosphorylation of MKK3 and -6, mitogen-activated protein kinase kinases (MKKs) regulated by MEKK4 in the p38 pathway. Phosphorylation of MAPK-activated protein kinase 2, a p38 substrate, is also inhibited, demonstrating a loss of p38 activity in MEKK4(K1361R) embryos. In contrast, the MEK1/2-extracellular signal-regulated kinase 1 (ERK1)/ERK2 and MKK4-Jun N-terminal protein kinase pathways were unaffected. The p38 pathway has been shown to regulate the phosphorylation and expression of the small heat shock protein HSP27. Compared to the wild type, MEKK4(K1361R) fibroblasts showed significantly reduced phosphorylation of p38 and HSP27, with a corresponding heat shock-induced instability of the actin cytoskeleton. Together, these data demonstrate MEKK4 regulation of p38 and that substrates downstream of p38 control cellular homeostasis. The findings are the first demonstration that MEKK4-regulated p38 activity is critical for neurulation.

Ablation of MEKK4 kinase activity causes neurulation and skeletal patterning defects in the mouse embryo.

B lymphoblastoid &#xB77C;&#xC774;&#xBE0C;&#xB7EC;&#xB9AC;&#xC5D0;&#xC11C; &#xBCF5;&#xC81C; &#xB41C; &#xBA54; &#xB974; &#xC218;&#xC6A9; &#xCCB4; &#xD2F0;&#xB85C;&#xC2E0; &#xD0A4; &#xCE58;&#xD0A8; retroviral oncogene v eyk&#xC758; &#xD3EC;&#xC720;&#xB958; orthologue &#xC774;&#xBA70; antiapoptotic &#xBC0F; &#xBCC0;&#xD615; &#xC2E0;&#xD638; &#xD65C;&#xC131;&#xD654; &#xB420; &#xB54C; &#xBCF4;&#xB0C5;&#xB2C8;&#xB2E4;. T &#xC138;&#xD3EC; &#xAE09;&#xC131; &#xB9BC;&#xD504; &#xAD6C;&#xC131; &#xBC31;&#xD608;&#xBCD1; (ALL)&#xC640; &#xC7A0;&#xC7AC;&#xC801;&#xC73C;&#xB85C; &#xBA54; &#xB974; &#xC2DD; &#xC18C;&#xC131; &#xC778;&#xC9C0; &#xD655;&#xC778; &#xD558;&#xB824;&#xBA74; leukemogenesis&#xC5D0;&#xC11C; &#xC911;&#xC694; &#xD55C; &#xC6B0;&#xB9AC; &#xBD84;&#xC11D; &#xBA54; &#xB974; &#xC2DD; &#xC815;&#xC0C1;&#xC801;&#xC778; &#xC778;&#xAC04;&#xC758; thymocytes&#xACFC; &#xC138;&#xD3EC;&#xC640; &#xC18C;&#xC544;&#xC5D0;&#xC11C; &#xBAA8;&#xB4E0; &#xD658;&#xC790; &#xC0D8;&#xD50C;.

Proto-oncogene &#xBA54; &#xB974; &#xC18C;&#xC544;&#xACFC; T &#xC138;&#xD3EC; &#xAE09;&#xC131; &#xB9BC;&#xD504; &#xAD6C;&#xC131; &#xBC31;&#xD608;&#xBCD1;&#xC5D0;&#xC11C;&#xC758; &#xC18C;&#xC131; &#xC2DD;.

El receptor de la tirosina quinasa Mer, clonado a partir de una biblioteca B-linfoblastoide, es el ort&#xF3;logo mam&#xED;fero del oncog&#xE9;n retroviral pollo v-EYK y env&#xED;a se&#xF1;ales antiapopt&#xF3;ticas y transformaci&#xF3;n cuando se activa. Para determinar si la expresi&#xF3;n de Mer es ect&#xF3;pico en las c&#xE9;lulas T leucemia linfobl&#xE1;stica aguda (LLA) y potencialmente importante de la leucemia, analizamos la expresi&#xF3;n-Mer en condiciones normales de timocitos y linfocitos humanos y en TODAS las muestras de pacientes pedi&#xE1;tricos.

La expresi&#xF3;n ect&#xF3;pica de la Mer en el proto-oncogen Pedi&#xE1;trica de c&#xE9;lulas T leucemia linfobl&#xE1;stica aguda

&#x4ECE; B &#x6C38;&#x751F;&#x5E93;&#xFF0C;&#x514B;&#x9686;&#x7684; Mer &#x53D7;&#x4F53;&#x916A;&#x6C28;&#x9178;&#x6FC0;&#x9176;&#x662F;&#x54FA;&#x4E73;&#x52A8;&#x7269;&#x7684;&#x9E21;&#x9006;&#x8F6C;&#x5F55;&#x75C5;&#x6BD2;&#x764C;&#x57FA;&#x56E0; v-eyk orthologue &#x5E76;&#x53D1;&#x9001;&#x6297;&#x7EC6;&#x80DE;&#x51CB;&#x4EA1;&#x548C;&#x6539;&#x53D8;&#x65F6;&#x6FC0;&#x6D3B;&#x7684;&#x4FE1;&#x53F7;&#x3002;&#x8981;&#x786E;&#x5B9A;&#x662F;&#x5426; Mer &#x8868;&#x8FBE;&#x5F0F;&#x662F;&#x5F02;&#x4F4D; T &#x7EC6;&#x80DE;&#x6025;&#x6027;&#x6DCB;&#x5DF4;&#x7EC6;&#x80DE;&#x6027;&#x767D;&#x8840;&#x75C5; (&#x5168;&#x90E8;) &#x548C;&#x6F5C;&#x5728;&#x91CD;&#x8981;&#x7684;&#x767D;&#x8840;&#x75C5;&#x7EC6;&#x80DE;&#xFF0C;&#x6211;&#x4EEC;&#x5206;&#x6790;&#x4E86; Mer &#x8868;&#x8FBE;&#x5728;&#x6B63;&#x5E38;&#x4EBA;&#x4F53;&#x80F8;&#x817A;&#x7EC6;&#x80DE;&#x548C;&#x6DCB;&#x5DF4;&#x7EC6;&#x80DE;&#x548C;&#x513F;&#x79D1;&#x6240;&#x6709;&#x60A3;&#x8005;&#x6837;&#x672C;&#x3002;

&#x539F;&#x764C;&#x57FA;&#x56E0; Mer &#x5C0F;&#x513F; T &#x7EC6;&#x80DE;&#x6025;&#x6027;&#x6DCB;&#x5DF4;&#x7EC6;&#x80DE;&#x767D;&#x8840;&#x75C5;&#x7684;&#x5F02;&#x4F4D;&#x8868;&#x8FBE;&#x3002;

The Mer receptor tyrosine kinase, cloned from a B-lymphoblastoid library, is the mammalian orthologue of the chicken retroviral oncogene v-eyk and sends antiapoptotic and transforming signals when activated. To determine if Mer expression is ectopic in T-cell acute lymphoblastic leukemia (ALL) and potentially important in leukemogenesis, we analyzed Mer expression in normal human thymocytes and lymphocytes and in pediatric ALL patient samples.

Ectopic expression of the proto-oncogene Mer in pediatric T-cell acute lymphoblastic leukemia.

La kinase Mer r&#xE9;cepteur de la tyrosine, clon&#xE9; &#xE0; partir d&#x26;#39;une biblioth&#xE8;que B-lymphoblasto&#xEF;des, est l&#x26;#39;orthologue mammif&#xE8;re de l&#x26;#39;oncog&#xE8;ne v-r&#xE9;troviral de poulet Eyk et envoie des signaux anti-apoptotiques et la transformation lorsqu&#x26;#39;il est activ&#xE9;. Pour d&#xE9;terminer si l&#x26;#39;expression ectopique Mer est en T-cellules de leuc&#xE9;mie lymphoblastique aigu&#xEB; (LLA) et potentiellement important dans la leuc&#xE9;mogen&#xE8;se, nous avons analys&#xE9; l&#x26;#39;expression Mer en thymocytes humains normaux et de lymphocytes et p&#xE9;diatriques Tous les &#xE9;chantillons des patients.

L&#x26;#39;expression ectopique de la Mer proto-oncog&#xE8;ne en p&#xE9;diatrie cellules T leuc&#xE9;mie aigu&#xEB; lymphoblastique

Mer-Tyrosinkinase, geklont aus einer B-Lymphoblastoidzellen-Bibliothek ist die S&auml;ugetier-Orthologue das Huhn retrovirale Onkogen V-Eyk und sendet Antiapoptotic und Umwandlung von Signalen, wenn aktiviert. Um festzustellen, ob Mer Ausdruck ektopische in T-Zelle akute lymphoblastische Leuk&auml;mie (ALL) und potenziell ist analysiert in Leukemogenesis wichtig, wir Mer Ausdruck in normalen menschlichen Thymozyten und Lymphozyten und in p&auml;diatrischen alle Patientenproben.

Ektopische Expression der Proto-oncogene Mer in p&auml;diatrische T-Zell-akute lymphoblastische Leuk&auml;mie.

La Mer recettore tirosina chinasi, clonata da una raccolta di B-linfoblastoidi, &egrave; il mammifero orthologue del pollo del oncogene retrovirale v-eyk e invia antiapoptotici e trasformando segnali quando attivato. Per determinare se Mer espressione ectopica in una T-cellula leucemia linfoblastica acuta (ALL) e potenzialmente importante nella leucemogenesi, abbiamo analizzato Mer espressione in linfociti e timociti umani normali e in pediatrica tutti i campioni dei pazienti.

Espressione ectopica della Mer proto-oncogene di leucemia linfoblastica acuta a cellule T pediatrica.

B-&#x30EA;&#x30F3;&#x30D1;&#x82BD;&#x7403;&#x30E9;&#x30A4;&#x30D6;&#x30E9;&#x30EA;&#x30FC;&#x304B;&#x3089;&#x30AF;&#x30ED;&#x30FC;&#x30CB;&#x30F3;&#x30B0;&#x3055;&#x308C;&#x305F;Mer&#x306E;&#x53D7;&#x5BB9;&#x4F53;&#x30C1;&#x30ED;&#x30B7;&#x30F3;&#x30AD;&#x30CA;&#x30FC;&#x30BC;&#x306F;&#x3001;&#x30CB;&#x30EF;&#x30C8;&#x30EA;&#x30EC;&#x30C8;&#x30ED;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x764C;&#x907A;&#x4F1D;&#x5B50;V-eyk&#x306E;&#x54FA;&#x4E73;&#x985E;&#x30AA;&#x30EB;&#x30BD;&#x30ED;&#x30B0;&#x3067;&#x3042;&#x308A;&#x3001;&#x6D3B;&#x6027;&#x5316;&#x3055;&#x308C;&#x308B;&#x3068;&#x30A2;&#x30DD;&#x30C8;&#x30FC;&#x30B7;&#x30B9;&#x3068;&#x5909;&#x63DB;&#x4FE1;&#x53F7;&#x3092;&#x9001;&#x4FE1;&#x3057;&#x307E;&#x3059;&#x3002; Mer&#x306E;&#x767A;&#x73FE;&#x306F;&#x3001;&#x767D;&#x8840;&#x75C5;&#x3067;T&#x7D30;&#x80DE;&#x6025;&#x6027;&#x30EA;&#x30F3;&#x30D1;&#x82BD;&#x7403;&#x6027;&#x767D;&#x8840;&#x75C5;&#xFF08;ALL&#xFF09;&#x306B;&#x304A;&#x3051;&#x308B;&#x7570;&#x6240;&#x6027;&#x3068;&#x6F5C;&#x5728;&#x7684;&#x306B;&#x91CD;&#x8981;&#x3067;&#x3042;&#x308B;&#x304B;&#x3069;&#x3046;&#x304B;&#x3092;&#x5224;&#x65AD;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x3001;&#x6211;&#x3005;&#x306F;&#x6B63;&#x5E38;&#x30D2;&#x30C8;&#x80F8;&#x817A;&#x7D30;&#x80DE;&#x3084;&#x30EA;&#x30F3;&#x30D1;&#x7403;&#x3067;&#x306F;&#x3001;&#x5C0F;&#x5150;ALL&#x60A3;&#x8005;&#x30B5;&#x30F3;&#x30D7;&#x30EB;&#x4E2D;&#x306E;Mer&#x306E;&#x767A;&#x73FE;&#x3092;&#x89E3;&#x6790;&#x3057;&#x305F;&#x3002;

&#x5C0F;&#x5150;T&#x7D30;&#x80DE;&#x6025;&#x6027;&#x30EA;&#x30F3;&#x30D1;&#x6027;&#x767D;&#x8840;&#x75C5;&#x306B;&#x304A;&#x3051;&#x308B;&#x30D7;&#x30ED;&#x30C8;&#x30AA;&#x30F3;&#x30B3;&#x30B8;&#x30FC;&#x30F3;Mer&#x306E;&#x7570;&#x6240;&#x6027;&#x767A;&#x73FE;

Membrane-bound receptors generate soluble ligand-binding domains either by proteolytic cleavage of the extracellular domain or alternative mRNA splicing yielding a secreted protein. Mertk (Mer) is in a receptor tyrosine kinase family with Axl and Tyro-3, and all 3 receptors share the Gas6 ligand. Mer regulates macrophage activation, promotes apoptotic cell engulfment, and supports platelet aggregation and clot stability in vivo. We have found that the membrane-bound Mer protein is cleaved in the extracellular domain via a metalloproteinase. The cleavage results in the production of a soluble Mer protein released in a constitutive manner from cultured cells. Significant amounts of the soluble Mer protein were also detected in human plasma, suggesting its physiologic relevance. Cleavage of Mer was enhanced by treatment with LPS and PMA and was specifically inhibited by a tumor necrosis factor alpha-converting enzyme metalloproteinase inhibitor. As a decoy receptor for Gas6, soluble Mer prevented Gas6-mediated stimulation of membrane-bound Mer. The inhibition of Gas6 activity by soluble Mer led to defective macrophage-mediated engulfment of apoptotic cells. Furthermore, soluble Mer decreased platelet aggregation in vitro and prevented fatal collagen/epinephrine-induced thromboembolism in mice, suggesting a potential therapeutic use for soluble Mer in the treatment of clotting disorders.

A soluble form of the Mer receptor tyrosine kinase inhibits macrophage clearance of apoptotic cells and platelet aggregation.

The MerTK plays several important roles in normal macrophage physiology, including regulation of cytokine secretion and clearance of apoptotic cells. Mer signaling in other cell types, including malignant cells that ectopically overexpress the RTK, leads to downstream prosurvival pathway activation. We explored the hypothesis that Mer has a prosurvival role in macrophages exposed to oxidative stress. H(2)O(2) treatment of peritoneal exudate murine macrophages and J774 cells rapidly stimulated Mer phosphorylation in a concentration-dependent manner. Mer phosphorylation was dependent on the ligand Gas6, as treatment with warfarin or MerFc (a fusion protein of the extracellular domain of Mer and the Fc portion of human Ig), inhibitors of Gas6 activity, blocked H(2)O(2)-mediated activation of Mer. Antiapoptotic signals including pAkt and pErk 1/2 were increased dramatically (threefold and 4.5-fold, respectively) in WT Mer-positive macrophages compared with Mer KO macrophages stimulated with H(2)O(2). In a consistent manner, Mer expression led to decreased cleavage of proapoptotic indicators PARP and Caspase-3. Furthermore, Mer provided up to twofold enhanced cellular survival to primary macrophages exposed to H(2)O(2). These data represent the first report of Mer activation in response to oxidative stress and demonstrate the ability of Mer RTK to promote macrophage survival in disease states that involve an oxidative stress environment.

Mer tyrosine kinase (MerTK) promotes macrophage survival following exposure to oxidative stress.

Ectopic Mer expression promotes pro-survival signaling and contributes to leukemogenesis and chemoresistance in childhood acute lymphoblastic leukemia (ALL). Consequently, Mer kinase inhibitors may promote leukemic cell death and further act as chemosensitizers increasing efficacy and reducing toxicities of current ALL regimens. We have applied a structure-based design approach to discover novel small molecule Mer kinase inhibitors. Several pyrazolopyrimidine derivatives effectively inhibit Mer kinase activity at sub-nanomolar concentrations. Furthermore, the lead compound shows a promising selectivity profile against a panel of 72 kinases and has excellent pharmacokinetic properties. We also describe the crystal structure of the complex between the lead compound and Mer, opening new opportunities for further optimization and new template design.

Discovery of Novel Small Molecule Mer Kinase Inhibitors for the Treatment of Pediatric Acute Lymphoblastic Leukemia.

Metastatic melanoma is one of the most aggressive forms of cutaneous cancers. Although recent therapeutic advances have prolonged patient survival, the prognosis remains dismal. C-MER proto-oncogene tyrosine kinase (MERTK) is a receptor tyrosine kinase with oncogenic properties that is often overexpressed or activated in various malignancies. Using both protein immunohistochemistry and microarray analyses, we demonstrate that MERTK expression correlates with disease progression. MERTK expression was highest in metastatic melanomas, followed by primary melanomas, while the lowest expression was observed in nevi. Additionally, over half of melanoma cell lines overexpressed MERTK compared with normal human melanocytes; however, overexpression did not correlate with mutations in BRAF or RAS. Stimulation of melanoma cells with the MERTK ligand GAS6 resulted in the activation of several downstream signaling pathways including MAPK/ERK, PI3K/AKT, and JAK/STAT. MERTK inhibition via shRNA reduced MERTK-mediated downstream signaling, reduced colony formation by up to 59%, and diminished tumor volume by 60% in a human melanoma murine xenograft model. Treatment of melanoma cells with UNC1062, a novel MERTK-selective small-molecule tyrosine kinase inhibitor, reduced activation of MERTK-mediated downstream signaling, induced apoptosis in culture, reduced colony formation in soft agar, and inhibited invasion of melanoma cells. This work establishes MERTK as a therapeutic target in melanoma and provides a rationale for the continued development of MERTK-targeted therapies.

MERTK receptor tyrosine kinase is a therapeutic target in melanoma.

Abnormal activation of Mer kinase has been implicated in the oncogenesis of many human cancers including acute lymphoblastic and myeloid leukemia, non-small cell lung cancer, and glioblastoma. We have discovered a new family of small molecule Mer inhibitors, pyrazolopyrimidine sulfonamides, that potently inhibit the kinase activity of Mer. Importantly, these compounds do not demonstrate significant hERG activity in the PatchXpress assay. Through structure-activity relationship studies, 35 (UNC1062) was identified as a potent (IC50 = 1.1 nM) and selective Mer inhibitor. When applied to live tumor cells, UNC1062 inhibited Mer phosphorylation and colony formation in soft agar. Given the potential of Mer as a therapeutic target, UNC1062 is a promising candidate for further drug development.

UNC1062, a new and potent Mer inhibitor.

Acute lymphoblastic leukemia (ALL) is the most common malignancy in children. Although survival rates have improved, patients with certain biological subtypes still have suboptimal outcomes. Current chemotherapeutic regimens are associated with short- and long-term toxicities and novel, less toxic therapeutic strategies are needed. Mer receptor tyrosine kinase is ectopically expressed in ALL patient samples and cell lines. Inhibition of Mer expression reduces pro-survival signaling, increases chemosensitivity, and delays development of leukemia in vivo suggesting that Mer tyrosine kinase inhibitors are excellent candidates for targeted therapies. Brain and spinal tumors are the second most common malignancies in childhood. Multiple chemotherapy approaches and radiation have been attempted, yet overall survival remains dismal. Mer is also abnormally expressed in atypical teratoid/rhabdoid tumors (ATRT), providing a rationale for targeting Mer as a therapeutic strategy. We have previously described UNC569, the first small molecule Mer inhibitor. This manuscript describes the biochemical and biological effects of UNC569 in ALL and ATRT. UNC569 inhibited Mer activation and downstream signaling through ERK1/2 and AKT, determined by western blot analysis. Treatment with UNC569 reduced proliferation/survival in liquid culture, decreased colony formation in methylcellulose/soft agar, and increased sensitivity to cytotoxic chemotherapies. MYC transgenic zebrafish with T-ALL were treated with UNC569 (4 Î¼M for 2 weeks). Fluorescence was quantified as indicator of the distribution of lymphoblasts, which express Mer and enhanced green fluorescent protein. UNC569 induced >50% reduction in tumor burden compared to vehicle- and mock-treated fish. These data support further development of Mer inhibitors as effective therapies in ALL and ATRT.

Susan Sather

Department of Pediatrics

University of Colorado Anschutz Medical Campus

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

Susan Sather

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Pediatrics

University of Colorado Anschutz Medical Campus

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

Department of Pediatrics