JAK inhibition decreases the autoimmune burden in Down syndrome.

Individuals with Down syndrome (DS), the genetic condition caused by trisomy 21 (T21), display clear signs of immune dysregulation, including high rates of autoimmune disorders and severe complications from infections. Although it is well established that T21 causes increased interferon responses and JAK/STAT signaling, elevated autoantibodies, global immune remodeling, and hypercytokinemia, the interplay between these processes, the clinical manifestations of DS, and potential therapeutic interventions remain ill defined. Here, we report a comprehensive analysis of immune dysregulation at the clinical, cellular, and molecular level in hundreds of individuals with DS. We demonstrate multi-organ autoimmunity of pediatric onset concurrent with unexpected autoantibody-phenotype associations. Importantly, constitutive immune remodeling and hypercytokinemia occur from an early age prior to autoimmune diagnoses or autoantibody production. We then report the interim analysis of a Phase II clinical trial investigating the safety and efficacy of the JAK inhibitor tofacitinib through multiple clinical and molecular endpoints. Analysis of the first 10 participants to complete the 16-week study shows a good safety profile and no serious adverse events. Treatment reduced skin pathology in alopecia areata, psoriasis, and atopic dermatitis, while decreasing interferon scores, cytokine scores, and levels of pathogenic autoantibodies without overt immune suppression. Additional research is needed to define the effects of JAK inhibition on the broader developmental and clinical hallmarks of DS. ClinicalTrials.gov identifier: NCT04246372.

Proteasome Inhibition Sensitizes Liposarcoma to MDM2 Inhibition with Nutlin-3 by Activating the ATF4/CHOP Stress Response Pathway.

Liposarcoma is the most commonly occurring soft-tissue sarcoma and is frequently characterized by amplification of chromosome region 12q13-15 harboring the oncogenes MDM2 and CDK4. This unique genetic profile makes liposarcoma an attractive candidate for targeted therapeutics. While CDK4/6 inhibitors are currently employed for treatment of several cancers, MDM2 inhibitors have yet to attain clinical approval. Here, we report the molecular characterization of the response of liposarcoma to the MDM2 inhibitor nutlin-3. Treatment with nutlin-3 led to upregulation of two nodes of the proteostasis network: the ribosome and the proteasome. CRISPR/Cas9 was used to perform a genome-wide loss of function screen that identified PSMD9, which encodes a proteasome subunit, as a regulator of response to nutlin-3. Accordingly, pharmacologic studies with a panel of proteasome inhibitors revealed strong combinatorial induction of apoptosis with nutlin-3. Mechanistic studies identified activation of the ATF4/CHOP stress response axis as a potential node of interaction between nutlin-3 and the proteasome inhibitor carfilzomib. CRISPR/Cas9 gene editing experiments confirmed that ATF4, CHOP, and the BH3-only protein, NOXA, are all required for nutlin-3 and carfilzomib-induced apoptosis. Furthermore, activation of the unfolded protein response using tunicamycin and thapsigargin was sufficient to activate the ATF4/CHOP stress response axis and sensitize to nutlin-3. Finally, cell line and patient-derived xenograft models demonstrated combinatorial effects of treatment with idasanutlin and carfilzomib on liposarcoma growth in vivo. Together, these data indicate that targeting of the proteasome could improve the efficacy of MDM2 inhibitors in liposarcoma. SIGNIFICANCE: Targeting the proteasome in combination with MDM2 inhibition activates the ATF4/CHOP stress response axis to induce apoptosis in liposarcoma, providing a potential therapeutic approach for the most common soft-tissue sarcoma.

MERTK inhibition alters the PD-1 axis and promotes anti-leukemia immunity.

MERTK is ectopically expressed and promotes survival in acute lymphoblastic leukemia (ALL) cells and is thus a potential therapeutic target. Here we demonstrate both direct therapeutic effects of MERTK inhibition on leukemia cells and induction of anti-leukemia immunity via suppression of the coinhibitory PD-1 axis. A MERTK-selective tyrosine kinase inhibitor, MRX-2843, mediated therapeutic anti-leukemia effects in immunocompromised mice bearing a MERTK-expressing human leukemia xenograft. In addition, inhibition of host MERTK by genetic deletion (Mertk-/- mice) or treatment with MRX-2843 significantly decreased tumor burden and prolonged survival in immune-competent mice inoculated with a MERTK-negative ALL, suggesting immune-mediated therapeutic activity. In this context, MERTK inhibition led to significant decreases in expression of the coinhibitory ligands PD-L1 and PD-L2 on CD11b+ monocytes/macrophages in the leukemia microenvironment. Furthermore, although T cells do not express MERTK, inhibition of MERTK indirectly decreased PD-1 expression on CD4+ and CD8+ T cells and decreased the incidence of splenic FOXP3+ Tregs at sites of leukemic infiltration, leading to increased T cell activation. These data demonstrate direct and immune-mediated therapeutic activities in response to MERTK inhibition in ALL models and provide validation of a translational agent targeting MERTK for modulation of tumor immunity.

CDK8 Kinase Activity Promotes Glycolysis.

Aerobic glycolysis, also known as the Warburg effect, is a hallmark of cancerous tissues. Despite its importance in cancer development, our understanding of mechanisms driving this form of metabolic reprogramming is incomplete. We report here an analysis of colorectal cancer cells engineered to carry a single point mutation in the active site of the Mediator-associated kinase CDK8, creating hypomorphic alleles sensitive to bulky ATP analogs. Transcriptome analysis revealed that CDK8 kinase activity is required for the expression of many components of the glycolytic cascade. CDK8 inhibition impairs glucose transporter expression, glucose uptake, glycolytic capacity and reserve, as well as cell proliferation and anchorage-independent growth, both in normoxia and hypoxia. Importantly, CDK8 impairment sensitizes cells to pharmacological glycolysis inhibition, a result reproduced with Senexin A, a dual inhibitor of CDK8/CDK19. Altogether, these results contribute to our understanding of CDK8 as an oncogene, and they justify investigations to target CDK8 in highly glycolytic tumors.

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.

Trisomy 21 consistently activates the interferon response.

Although it is clear that trisomy 21 causes Down syndrome, the molecular events acting downstream of the trisomy remain ill defined. Using complementary genomics analyses, we identified the interferon pathway as the major signaling cascade consistently activated by trisomy 21 in human cells. Transcriptome analysis revealed that trisomy 21 activates the interferon transcriptional response in fibroblast and lymphoblastoid cell lines, as well as circulating monocytes and T cells. Trisomy 21 cells show increased induction of interferon-stimulated genes and decreased expression of ribosomal proteins and translation factors. An shRNA screen determined that the interferon-activated kinases JAK1 and TYK2 suppress proliferation of trisomy 21 fibroblasts, and this defect is rescued by pharmacological JAK inhibition. Therefore, we propose that interferon activation, likely via increased gene dosage of the four interferon receptors encoded on chromosome 21, contributes to many of the clinical impacts of trisomy 21, and that interferon antagonists could have therapeutic benefits.

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.

Tyrosine kinase inhibition in leukemia induces an altered metabolic state sensitive to mitochondrial perturbations.

PURPOSE: Although tyrosine kinase inhibitors (TKI) can be effective therapies for leukemia, they fail to fully eliminate leukemic cells and achieve durable remissions for many patients with advanced BCR-ABL(+) leukemias or acute myelogenous leukemia (AML). Through a large-scale synthetic lethal RNAi screen, we identified pyruvate dehydrogenase, the limiting enzyme for pyruvate entry into the mitochondrial tricarboxylic acid cycle, as critical for the survival of chronic myelogenous leukemia (CML) cells upon BCR-ABL inhibition. Here, we examined the role of mitochondrial metabolism in the survival of Ph(+) leukemia and AML upon TK inhibition. EXPERIMENTAL DESIGN: Ph(+) cancer cell lines, AML cell lines, leukemia xenografts, cord blood, and patient samples were examined. RESULTS: We showed that the mitochondrial ATP-synthase inhibitor oligomycin-A greatly sensitized leukemia cells to TKI in vitro. Surprisingly, oligomycin-A sensitized leukemia cells to BCR-ABL inhibition at concentrations of 100- to 1,000-fold below those required for inhibition of respiration. Oligomycin-A treatment rapidly led to mitochondrial membrane depolarization and reduced ATP levels, and promoted superoxide production and leukemia cell apoptosis when combined with TKI. Importantly, oligomycin-A enhanced elimination of BCR-ABL(+) leukemia cells by TKI in a mouse model and in primary blast crisis CML samples. Moreover, oligomycin-A also greatly potentiated the elimination of FLT3-dependent AML cells when combined with an FLT3 TKI, both in vitro and in vivo. CONCLUSIONS: TKI therapy in leukemia cells creates a novel metabolic state that is highly sensitive to particular mitochondrial perturbations. Targeting mitochondrial metabolism as an adjuvant therapy could therefore improve therapeutic responses to TKI for patients with BCR-ABL(+) and FLT3(ITD) leukemias.