Germline ETV6 mutation promotes inflammation and disrupts lymphoid development of early hematopoietic progenitors.

Germline mutations in ETV6 are associated with a syndrome of thrombocytopenia and leukemia predisposition, and ETV6 is among the most commonly mutated genes in leukemias, especially childhood B-cell acute lymphoblastic leukemia. However, the mechanisms underlying disease caused by ETV6 dysfunction are poorly understood. To address these gaps in knowledge, using CRISPR/Cas9, we developed a mouse model of the most common recurrent, disease-causing germline mutation in ETV6. We found defects in hematopoiesis related primarily to abnormalities of the multipotent progenitor population 4 (MPP4) subset of hematopoietic progenitor cells and evidence of sterile inflammation. Expression of ETV6 in Ba/F3 cells altered the expression of several cytokines, some of which were also detected at higher levels in the bone marrow of the mice with Etv6 mutation. Among these, interleukin-18 and interleukin-13 abrogated B-cell development of sorted MPP4 cells, but not common lymphoid progenitors, suggesting that inflammation contributes to abnormal hematopoiesis by impairing lymphoid development. These data, along with those from humans, support a model in which ETV6 dysfunction promotes inflammation, which adversely affects thrombopoiesis and promotes leukemogenesis.

ETV6 germline mutations cause HDAC3/NCOR2 mislocalization and upregulation of interferon response genes.

ETV6 is an ETS family transcription factor that plays a key role in hematopoiesis and megakaryocyte development. Our group and others have identified germline mutations in ETV6 resulting in autosomal dominant thrombocytopenia and predisposition to malignancy; however, molecular mechanisms defining the role of ETV6 in megakaryocyte development have not been well established. Using a combination of molecular, biochemical, and sequencing approaches in patient-derived PBMCs, we demonstrate abnormal cytoplasmic localization of ETV6 and the HDAC3/NCOR2 repressor complex that led to overexpression of HDAC3-regulated interferon response genes. This transcriptional dysregulation was also reflected in patient-derived platelet transcripts and drove aberrant proplatelet formation in megakaryocytes. Our results suggest that aberrant transcription may predispose patients with ETV6 mutations to bone marrow inflammation, dysplasia, and megakaryocyte dysfunction.

Pro-inflammatory cytokine blockade attenuates myeloid expansion in a murine model of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a debilitating autoimmune disease characterized by chronic inflammation and progressive destruction of joint tissue. It is also characterized by aberrant blood phenotypes including anemia and suppressed lymphopoiesis that contribute to morbidity in RA patients. However, the impact of RA on hematopoietic stem cells (HSC) has not been fully elucidated. Using a collagen-induced mouse model of human RA, we identified systemic inflammation and myeloid overproduction associated with activation of a myeloid differentiation gene program in HSC. Surprisingly, despite ongoing inflammation, HSC from arthritic mice remain in a quiescent state associated with activation of a proliferation arrest gene program. Strikingly, we found that inflammatory cytokine blockade using the interleukin-1 receptor antagonist anakinra led to an attenuation of inflammatory arthritis and myeloid expansion in the bone marrow of arthritic mice. In addition, anakinra reduced expression of inflammation-driven myeloid lineage and proliferation arrest gene programs in HSC of arthritic mice. Altogether, our findings show that inflammatory cytokine blockade can contribute to normalization of hematopoiesis in the context of chronic autoimmune arthritis.

Recovery and analysis of transcriptome subsets from pooled single-cell RNA-seq libraries.

Single-cell RNA sequencing (scRNA-seq) methods generate sparse gene expression profiles for thousands of single cells in a single experiment. The information in these profiles is sufficient to classify cell types by distinct expression patterns but the high complexity of scRNA-seq libraries often prevents full characterization of transcriptomes from individual cells. To extract more focused gene expression information from scRNA-seq libraries, we developed a strategy to physically recover the DNA molecules comprising transcriptome subsets, enabling deeper interrogation of the isolated molecules by another round of DNA sequencing. We applied the method in cell-centric and gene-centric modes to isolate cDNA fragments from scRNA-seq libraries. First, we resampled the transcriptomes of rare, single megakaryocytes from a complex mixture of lymphocytes and analyzed them in a second round of DNA sequencing, yielding up to 20-fold greater sequencing depth per cell and increasing the number of genes detected per cell from a median of 1313 to 2002. We similarly isolated mRNAs from targeted T cells to improve the reconstruction of their VDJ-rearranged immune receptor mRNAs. Second, we isolated CD3D mRNA fragments expressed across cells in a scRNA-seq library prepared from a clonal T cell line, increasing the number of cells with detected CD3D expression from 59.7% to 100%. Transcriptome resampling is a general approach to recover targeted gene expression information from single-cell RNA sequencing libraries that enhances the utility of these costly experiments, and may be applicable to the targeted recovery of molecules from other single-cell assays.

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% to 50% of acute lymphoblastic leukemias (ALL) and more than 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: Preclinical in vitro and in vivo assays using cell lines and primary leukemia patient samples were used to evaluate antileukemic effects of UNC2025.Results: UNC2025 potently inhibited prosurvival 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. In addition, 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, supports continued development of MERTK inhibitors for treatment of leukemia. Clin Cancer Res; 23(6); 1481-92. ©2016 AACR.

The MERTK/FLT3 inhibitor MRX-2843 overcomes resistance-conferring FLT3 mutations in acute myeloid leukemia.

FMS-like tyrosine kinase 3-targeted (FLT3-targeted) therapies have shown initial promise for the treatment of acute myeloid leukemia (AML) expressing FLT3-activating mutations; however, resistance emerges rapidly. Furthermore, limited options exist for the treatment of FLT3-independent AML, demonstrating the need for novel therapies that reduce toxicity and improve survival. MERTK receptor tyrosine kinase is overexpressed in 80% to 90% of AMLs and contributes to leukemogenesis. Here, we describe MRX-2843, a type 1 small-molecule tyrosine kinase inhibitor that abrogates activation of both MERTK and FLT3 and their downstream effectors. MRX-2843 treatment induces apoptosis and inhibits colony formation in AML cell lines and primary patient samples expressing MERTK and/or FLT3-ITD, with a wide therapeutic window compared with that of normal human cord blood cells. In murine orthotopic xenograft models, once-daily oral therapy prolonged survival 2- to 3-fold over that of vehicle-treated controls. Additionally, MRX-2843 retained activity against quizartinib-resistant FLT3-ITD-mutant proteins with clinically relevant alterations at the D835 or F691 loci and prolonged survival in xenograft models of quizartinib-resistant AML. Together, these observations validate MRX-2843 as a translational agent and support its clinical development for the treatment of AML.

Small Molecule Inhibition of MERTK Is Efficacious in Non-Small Cell Lung Cancer Models Independent of Driver Oncogene Status.

Treatment of non-small cell lung cancer (NSCLC) has been transformed by targeted therapies directed against molecular aberrations specifically activated within an individual patient's tumor. However, such therapies are currently only available against a small number of such aberrations, and new targets and therapeutics are needed. Our laboratory has previously identified the MERTK receptor tyrosine kinase (RTK) as a potential drug target in multiple cancer types, including NSCLC. We have recently developed UNC2025--the first-in-class small molecule inhibitor targeting MERTK with pharmacokinetic properties sufficient for clinical translation. Here, we utilize this compound to further validate the important emerging biologic functions of MERTK in lung cancer pathogenesis, to establish that MERTK can be effectively targeted by a clinically translatable agent, and to demonstrate that inhibition of MERTK is a valid treatment strategy in a wide variety of NSCLC lines independent of their driver oncogene status, including in lines with an EGFR mutation, a KRAS/NRAS mutation, an RTK fusion, or another or unknown driver oncogene. Biochemically, we report the selectivity of UNC2025 for MERTK, and its inhibition of oncogenic downstream signaling. Functionally, we demonstrate that UNC2025 induces apoptosis of MERTK-dependent NSCLC cell lines, while decreasing colony formation in vitro and tumor xenograft growth in vivo in murine models. These findings provide further evidence for the importance of MERTK in NSCLC, and demonstrate that MERTK inhibition by UNC2025 is a feasible, clinically relevant treatment strategy in a wide variety of NSCLC subtypes, which warrants further investigation in clinical trials.

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.

Mer590, a novel monoclonal antibody targeting MER receptor tyrosine kinase, decreases colony formation and increases chemosensitivity in non-small cell lung cancer.

The successes of targeted therapeutics against EGFR and ALK in non-small cell lung cancer (NSCLC) have demonstrated the substantial survival gains made possible by precision therapy. However, the majority of patients do not have tumors with genetic alterations responsive to these therapies, and therefore identification of new targets is needed. Our laboratory previously identified MER receptor tyrosine kinase as one such potential target. We now report our findings targeting MER with a clinically translatable agent--Mer590, a monoclonal antibody specific for MER. Mer590 rapidly and robustly reduced surface and total MER levels in multiple cell lines. Treatment reduced surface MER levels by 87%, and this effect was maximal within four hours. Total MER levels were also dramatically reduced, and this persisted for at least seven days. Mechanistically, MER down-regulation was mediated by receptor internalization and degradation, leading to inhibition of downstream signaling through STAT6, AKT, and ERK1/2. Functionally, this resulted in increased apoptosis, increased chemosensitivity to carboplatin, and decreased colony formation. In addition to carboplatin, Mer590 interacted cooperatively with shRNA-mediated MER inhibition to augment apoptosis. These data demonstrate that MER inhibition can be achieved with a monoclonal antibody in NSCLC. Optimization toward a clinically available anti-MER antibody is warranted.

Pseudo-cyclization through intramolecular hydrogen bond enables discovery of pyridine substituted pyrimidines as new Mer kinase inhibitors.

Abnormal activation or overexpression of Mer receptor tyrosine kinase has been implicated in survival signaling and chemoresistance in many human cancers. Consequently, Mer is a promising novel cancer therapeutic target. A structure-based drug design approach using a pseudo-ring replacement strategy was developed and validated to discover a new family of pyridinepyrimidine analogues as potent Mer inhibitors. Through SAR studies, 10 (UNC2250) was identified as the lead compound for further investigation based on high selectivity against other kinases and good pharmacokinetic properties. When applied to live cells, 10 inhibited steady-state phosphorylation of endogenous Mer with an IC50 of 9.8 nM and blocked ligand-stimulated activation of a chimeric EGFR-Mer protein. Treatment with 10 also resulted in decreased colony-forming potential in rhabdoid and NSCLC tumor cells, thereby demonstrating functional antitumor activity. The results provide a rationale for further investigation of this compound for therapeutic application in patients with cancer.