The JAK/STAT pathway is the primary signaling mechanism for a wide array of cytokines and growth factors, and mutations in JAK signaling have been implicated in the development of Cutaneous T-cell lymphomas (CTCLs). Elevated levels of phospho-STAT3 and activating mutations in the JAK/STAT pathway have been found in most CTCL cell lines and clinical samples, demonstrating that constitutive activation and dysregulation of the JAK/STAT pathway may play a role in CTCL pathogenesis. The JAK/STAT pathway is a highly conserved, pleiotropic cascade involved in a multitude of signal transduction pathways in cell proliferation, differentiation, cell migration, and apoptosis. We tested 6 JAK inhibitors - baricitinib, momelotinib, ruxolitinib, tofacitinib, FM-381, and PF-06651600 – for their in vitro activity against 5 established CTCL cell lines. We observed significant differences in selective cytotoxicity among the agents, with the agents possessing potent JAK1 inhibitory function showing the most consistent cell killing in CTCL cell lines. Using MTT colorimetric assays, we found that baricitinib, momelotinib, and ruxolitinib demonstrate potent in vitro cytotoxicity. We show that incubation with these drugs decreased JAK/STAT signaling using Western Blot analysis of phosphorylated and unphosphorylated STAT 1, 3, and 5. In summary, we show that targeting the JAK/STAT pathway may be a promising avenue for drug therapy in CTCL.

Cutaneous T-cell lymphoma (CTCL) is a clinically and genetically heterogeneous disease characterized by the infiltration of malignant CD4+ T lymphocytes in the skin. Mechlorethamine (nitrogen mustard, NM), an alkylating agent, and romidepsin, a histone deacetylase inhibitor, are two FDA-approved monotherapies for CTCL. In vitro analysis showed synergism of the drug combination in CTCL cell lines and primary samples from patients with Sézary Syndrome. We performed next generation RNA sequencing to elucidate the mechanism underlying the effects of this drug combination in CTCL. Three CTCL cell lines and CD4+ cells cultured from six primary samples were each treated with vehicle, 0.25μM NM, 1nM romidepsin, and the two drugs combined for 24 hours. Total RNA was extracted from the treated cells and was sequenced on an Illumina HiSeq 2500. Differential expression analysis, principal component analysis, KEGG pathway analysis, and gene set enrichment analysis (GSEA) were performed. We found a significantly larger overlap of genes enriched by romidepsin and the combination compared to NM and the combination (p<0.00001). A number of KEGG pathways were uniquely enriched with combination therapy compared to monotherapy, most notably downregulation of the JAK/STAT pathway in the cell lines. This result was supported by GSEA of primary samples, which showed significant downregulation of genes downregulated by JAK inhibitors and shRNA-JAK2 knockdown, and downregulation of genes upregulated by IL2 treatment. Constitutive activation of the JAK/STAT pathway has been identified previously in CTCL and our gene expression profiling data identifies this pathway as a promising target of the NM-romidepsin combination.

Combination regimens are the mainstay of treatment for hematologic malignancies, but there are currently no FDA-approved combination therapies for cutaneous T-cell lymphoma (CTCL), and few combinations have been studied systematically in vitro. We hypothesize that romidepsin, an HDAC inhibitor, allows a more open chromatin structure that can provide better access to the alkylating effects of mechlorethamine in malignant T cells. We have previously reported more than additive effects of romidepsin and mechlorethamine. Here, we formally evaluated synergy of these two drugs by examining their combined effect in 4 CTCL cell lines, SeAx, HH, Hut78, and Hut102, and 6 primary samples from patients with Sézary Syndrome. Single-agent dose response curves were generated using the cell lines to determine the approximate IC50 for each drug at 24, 48, 72, and 96 hours. The cell lines were treated at fixed ratios of the drugs using Chou and Talalay's median effect method to calculate isobolograms and combination indices (CI) to determine if the drugs are synergistic. Flow cytometry for apoptosis and cell viability was also performed to characterize the combined effects of the drug compared to single agents and controls. Our results showed synergism of romidepsin and mechlorethamine in all 4 cell lines, and 5 of 6 patient samples showed enhanced cytotoxic effects of the combination in malignant cells compared to single agents. The synergistic effects of romidepsin and mechlorethamine in CTCL/Sézary Syndrome make a strong argument to test this drug combination in clinical trials.

The TAL family of transcription factors consists of the DNA-binding proteins TAL1, TAL2, and LYL1 and forms a specific subtype within the larger group of basic helix-loop-helix (bHLH) proteins. Here, we discuss the role of these TAL proteins in normal development and highlight their proposed oncogenic function in the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL).

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy resulting from leukemic transformation of T-cell progenitors in the thymus. It accounts for approximately 15% of ALL cases in childhood and 20–25% in adults and is a leading cause of death in children. IL-7 and its receptor (IL-7R) play a critical role in normal T-cell development and homeostasis. The IL-7R gene was sequenced in T-ALL from three cohorts. Mutations in IL-7R were identified in 9% of pediatric T-ALL patients. These mutations usually involved insertions of three amino acids including cysteine and proline in the extracellular juxtamembrane region. WT or mutant forms of the human IL-7R (hIL-7R) from patients were retrovirally transfected into an IL-7-dependent murine thymic cell line D1. Mutant hIL-7Rs induced ligand-independent activation of the Jak-Stat and PI3K pathways, cell survival and proliferation. Notably, mutant hIL-7R-expressing D1 cells induced subcutaneous tumors in Rag1-/- mice, with substantial infiltration into various organs that are normally affected in advanced stages of T-ALL, such as bone marrow, liver, lymph nodes and spleen. Further functional assays revealed that mutant hIL-7Rs constitutive signaling required homodimerization via cysteines in the inserted sequences and downstream Jak1 activation, and was IL-7, gc and Jak3-independent. Our findings indicate that hIL-7R mutational activation drives T-ALL leukemogenesis and implicate IL-7R and Jak1 as therapeutic targets in T-ALL.

In T-cell acute lymphoblastic leukemia (T-ALL), transcription factors are known to be deregulated by chromosomal translocations, but mutations in protein tyrosine kinases have only rarely been identified 1 , 2 , 3 . Here we describe the extrachromosomal (episomal) 4 amplification of ABL1 in 5 of 90 (5.6%) individuals with T-ALL, an aberration that is not detectable by conventional cytogenetics. Molecular analyses delineated the amplicon as a 500-kb region from chromosome band 9q34, containing the oncogenes ABL1 and NUP214 (refs. 5 , 6 ). We identified a previously undescribed mechanism for activation of tyrosine kinases in cancer: the formation of episomes resulting in a fusion between NUP214 and ABL1 . We detected the NUP214-ABL1 transcript in five individuals with the ABL1 amplification, in 5 of 85 (5.8%) additional individuals with T-ALL and in 3 of 22 T-ALL cell lines. The constitutively phosphorylated tyrosine kinase NUP214-ABL1 is sensitive to the tyrosine kinase inhibitor imatinib 7 , 8 . The recurrent cryptic NUP214-ABL1 rearrangement is associated with increased HOX expression 1 and deletion of CDKN2A 9 , consistent with a multistep pathogenesis of T-ALL. NUP214-ABL1 expression defines a new subgroup of individuals with T-ALL who could benefit from treatment with imatinib.