UNC2025, a MERTK Small-Molecule Inhibitor, Is Therapeutically Effective Alone and in Combination with Methotrexate in Leukemia Models

MERTK tyrosine kinase is ectopically expressed in 30–50% of acute lymphoblastic leukemias (ALL) and over 80% of acute myeloid leukemias (AML) and is a potential therapeutic target. Here, we evaluated the utility of UNC2025, a MERTK tyrosine kinase inhibitor, for treatment of acute leukemia

Efficacy of a Mer and Flt3 tyrosine kinase small molecule inhibitor, UNC1666, in acute myeloid leukemia

Mer and Flt3 receptor tyrosine kinases have been implicated as therapeutic targets in acute myeloid leukemia (AML). In this manuscript we describe UNC1666, a novel ATP-competitive small molecule tyrosine kinase inhibitor, which potently diminishes Mer and Flt3 phosphorylation in AML. Treatment with UNC1666 mediated biochemical and functional effects in AML cell lines expressing Mer or Flt3 internal tandem duplication (ITD), including decreased phosphorylation of Mer, Flt3 and downstream effectors Stat, Akt and Erk, induction of apoptosis in up to 98% of cells, and reduction of colony formation by greater than 90%, compared to treatment with vehicle. These effects were dose-dependent, with inhibition of downstream signaling and functional effects correlating with the degree of Mer or Flt3 kinase inhibition. Treatment of primary AML patient samples expressing Mer and/or Flt3-ITD with UNC1666 also inhibited Mer and Flt3 intracellular signaling, induced apoptosis, and inhibited colony formation. In summary, UNC1666 is a novel potent small molecule tyrosine kinase inhibitor that decreases oncogenic signaling and myeloblast survival, thereby validating dual Mer/Flt3 inhibition as an attractive treatment strategy for AML

Germline mutations in ETV6 are associated with thrombocytopenia, red cell macrocytosis and predisposition to lymphoblastic leukemia

Some familial platelet disorders are associated with predisposition to leukemia, myelodysplastic syndrome (MDS) or dyserythropoietic anemia. We identified a family with autosomal dominant thrombocytopenia, high erythrocyte mean corpuscular volume (MCV) and two occurrences of B cell-precursor acute lymphoblastic leukemia (ALL). Whole-exome sequencing identified a heterozygous single-nucleotide change in ETV6 (ets variant 6), c.641C>T, encoding a p.Pro214Leu substitution in the central domain, segregating with thrombocytopenia and elevated MCV. A screen of 23 families with similar phenotypes identified 2 with ETV6 mutations. One family also had a mutation encoding p.Pro214Leu and one individual with ALL. The other family had a c.1252A>G transition producing a p.Arg418Gly substitution in the DNA-binding domain, with alternative splicing and exon skipping. Functional characterization of these mutations showed aberrant cellular localization of mutant and endogenous ETV6, decreased transcriptional repression and altered megakaryocyte maturation. Our findings underscore a key role for ETV6 in platelet formation and leukemia predisposition

Promoter Specificity and Efficacy in Conditional and Inducible Transgenic Targeting of Lung Macrophages

Conditional and inducible Cre-loxP systems are used to target gene deletion to specific cell lineages and tissues through promoter-restricted expression of the bacterial DNA recombinase, Cre. Although Cre-loxP systems are widely used to target gene deletion in lung macrophages, limited data are published on the specificity and efficiency of “macrophage targeting” Cre lines. Using R26-stopfl/fl-TdTomato and tetOn-GFP reporter lines, we assessed the specificity and efficiency of four commercially available Cre driver lines that are often considered “macrophage specific.” We evaluated two conditional (Csf1r-Cre and LysM-Cre) and two inducible [CX3CR1-estrogen receptor-Cre (ERCre) and CD68-rtTA] lines. We assessed Cre activation in six resident lung myeloid populations, as well as activation in lung leukocytes, lung epithelial and endothelial cells, peripheral blood leukocytes, and tissue macrophages of the spleen, bone marrow, and peritoneal cavity. Although Csf1r-Cre and LysM-Cre target resident alveolar macrophages (ResAM) and interstitial macrophages (IM) with high efficiency, neither line is specific for macrophages. Csf1r-Cre targets all leukocyte populations, while LysM-Cre targets dendritic cell, neutrophils, monocytes, and a quarter of lung epithelial cells. CX3CR1-ERCre and CD68-rtTA both target IM, but do not target ResAM. Further, although neither line is specific for macrophages, a pulse-wait administration of tamoxifen or doxycycline can be used to significantly improve IM specificity in these inducible lines. In summary, while Cre-loxP remains a powerful tool to study macrophage function, numerous pitfalls exist. Herein, we document strengths and weaknesses of Csf1r-Cre, LysM-Cre, CX3CR1-ERCre, and CD68-rtTA systems for targeting specific macrophage populations in the lungs and provide data that will aid investigators in selecting the proper strain

DataSheet_1_Serum lactate is associated with increased illness severity in immunocompromised pediatric hematology oncology patients presenting to the emergency department with fever.docx

IntroductionDetermining which febrile pediatric hematology/oncology (PHO) patients will decompensate from severe infection is a significant challenge. Serum lactate is a well-established marker of illness severity in general adult and pediatric populations, however its utility in PHO patients is unclear given that chemotherapy, organ dysfunction, and cancer itself can alter lactate metabolism. In this retrospective analysis, we studied the association of initial serum lactate in febrile immunosuppressed PHO patients with illness severity, defined by the incidence of clinical deterioration events (CDE) and invasive bacterial infection (IBI) within 48 hours.MethodsReceiver operating characteristic (ROC) curves were reported using initial lactate within two hours of arrival as the sole predictor for CDE and IBI within 48 hours. Using a generalized estimating equations (GEE) approach, the association of lactate with CDE and IBI within 48 hours was tested in univariate and multivariable analyses including covariates based on Quasi-likelihood under Independence Model Criterion (QIC). Additionally, the association of lactate with secondary outcomes (i.e., hospital length of stay (LOS), intensive care unit (PICU) admission, PICU LOS, non-invasive infection) was assessed.ResultsAmong 897 encounters, 48 encounters had ≥1 CDE (5%), and 96 had ≥1 IBI (11%) within 48 hours. Elevated lactate was associated with increased CDE in univariate (OR 1.77, 95%CI: 1.48-2.12, pConclusionsThese data demonstrated an independent association of elevated initial lactate level and increased illness severity in febrile PHO patients, suggesting that serum lactate could be incorporated into future risk stratification strategies for this population.</p

UNC2025, a MERTK Small-Molecule Inhibitor, Is Therapeutically Effective Alone and in Combination with Methotrexate in Leukemia Models

PURPOSE: MERTK tyrosine kinase is ectopically expressed in 30–50% of acute lymphoblastic leukemias (ALL) and over 80% of acute myeloid leukemias (AML) and is a potential therapeutic target. Here, we evaluated the utility of UNC2025, a MERTK tyrosine kinase inhibitor, for treatment of acute leukemia. EXPERIMENTAL DESIGN: Pre-clinical in vitro and in vivo assays using cell lines and primary leukemia patient samples were utilized to evaluate anti-leukemic effect of UNC2025. RESULTS: UNC2025 potently inhibited pro-survival signaling, induced apoptosis and reduced proliferation and colony formation in MERTK-expressing ALL and AML cell lines and patient samples. Approximately 30% of primary leukemia patient samples (78 of 261 total) were sensitive to UNC2025. Sensitive samples were most prevalent in the AML, T-ALL, and minimally differentiated (M0) AML subsets. UNC2025 inhibited MERTK in bone marrow leukemia cells and had significant therapeutic effects in xenograft models, with dose-dependent decreases in tumor burden and consistent two-fold increases in median survival, irrespective of starting disease burden. In a patient-derived AML xenograft model, treatment with UNC2025 induced disease regression. Additionally, UNC2025 increased sensitivity to methotrexate in vivo, suggesting that addition of MERTK-targeted therapy to current cytotoxic regimens may be particularly effective and/or allow for chemotherapy dose reduction. CONCLUSIONS: The broad spectrum activity mediated by UNC2025 in leukemia patient samples and xenograft models, alone or in combination with cytotoxic chemotherapy, support continued development of MERTK inhibitors for treatment of leukemia

MERTK Inhibition Induces Polyploidy and Promotes Cell Death and Cellular Senescence in Glioblastoma Multiforme.

MER receptor tyrosine kinase (MERTK) is expressed in a variety of malignancies, including glioblastoma multiforme (GBM). Our previous work demonstrated that inhibition of MERTK using RNA interference induced cell death and chemosensitivity in GBM cells, implicating MERTK as a potential therapeutic target. Here we investigate whether a novel MERTK-selective small molecule tyrosine kinase inhibitor, UNC2025, has similar anti-tumor effects in GBM cell lines.Correlations between expression of GAS6, a MERTK ligand, and prognosis were determined using data from the TCGA database. GBM cell lines (A172, SF188, U251) were treated in vitro with increasing doses of UNC2025 (50-400nM). Cell count and viability were determined by trypan blue exclusion. Cell cycle profiles and induction of apoptosis were assessed by flow cytometric analysis after BrdU or Po-Pro-1/propidium iodide staining, respectively. Polyploidy was detected by propidium iodide staining and metaphase spread. Cellular senescence was determined by β-galactosidase staining and senescence-associated secretory cytokine analysis.Decreased overall survival significantly correlated with high levels of GAS6 expression in GBM, highlighting the importance of TAM kinase signaling in GBM tumorigenesis and/or therapy resistance and providing strong rationale for targeting these pathways in the clinic. All three GBM cell lines exhibited dose dependent reductions in cell number and colony formation (>90% at 200nM) after treatment with UNC2025. Cell cycle analysis demonstrated accumulation of cells in the G2/M phase and development of polyploidy. After extended exposure, 60-80% of cells underwent apoptosis. The majority of surviving cells (65-95%) were senescent and did not recover after drug removal. Thus, UNC2025 mediates anti-tumor activity in GBM by multiple mechanisms.The findings described here provide further evidence of oncogenic roles for MERTK in GBM, demonstrate the importance of kinase activity for MERTK tumorigenicity and validate UNC2025, a novel MERTK inhibitor, as a potential therapeutic agent for treatment of GBM