Uncoupling JAK3 activation induces apoptosis in human lymphoid cancer cells via regulating critical survival pathways

AbstractIn the current work, we report that specific inhibition of Janus tyrosine kinase (JAK3) via NC1153 induces apoptosis of certain leukemia/lymphoma cell lines. Affymetrix microarray profiling following NC1153 treatment unveiled JAK3 dependent survival modulating pathways (p53, TGF-β, TNFR and ER stress) in Kit225 cells. IL-2 responsive NC1153 target genes were regulated in human JAK3 positive, but not in JAK3 negative lymphoid tumor cells. Moreover, primary lymphoma samples revealed that a number of these genes were reciprocally regulated during disease progression and JAK3 inhibition suggesting that downstream targets of JAK3 could be exploited in the development of novel cancer treatment regimes

Genome wide mapping reveals PDE4B as an IL-2 induced STAT5 target gene in activated human PBMCs and lymphoid cancer cells

IL-2 is the primary growth factor for promoting survival and proliferation of activated T cells that occurs following engagement of the Janus Kinase (JAK)1-3/and Signal Transducer and Activator of Transcription (STAT) 5 signaling pathway. STAT5 has two isoforms: STAT5A and STAT5B ( commonly referred to as STAT5) which, in T cells, play redundant roles transcribing cell cycle and survival genes. As such, inhibition of STAT5 by a variety of mechanisms can rapidly induce apoptosis in certain lymphoid tumor cells, suggesting that it and its target genes represent therapeutic targets to control certain lymphoid diseases. To search for these molecules we aligned IL-2 regulated genes detected by Affymetrix gene expression microarrays with the STAT5 cistrome identified by chip-on-ChIP analysis in an IL-2-dependent human leukemia cell line, Kit225. Select overlapping genes were then validated using qRT(2)PCR medium-throughput arrays in human PHA-activated PBMCs. Of 19 putative genes, one key regulator of T cell receptor signaling, PDE4B, was identified as a novel target, which was readily up-regulated at the protein level (3 h) in IL-2 stimulated, activated human PBMCs. Surprisingly, only purified CD8+ primary T-cells expressed PDE4B, but not CD4+ cells. Moreover, PDE4B was found to be highly expressed in CD4+ lymphoid cancer cells, which suggests that it may represent a physiological role unique to the CD8+ and lymphoid cancer cells and thus might represent a target for pharmaceutical intervention for certain lymphoid diseases

cAMP regulates IL-2 receptor signaling in human T cells

Proper immune system function is dependent on positive and negative regulation of T cell signaling pathways. Full T cell activation requires sequential signaling through the T cell receptor (TCR), costimulatory molecules and the IL-2 receptor (IL-2R). The IL-2R associated Janus tyrosine kinase 3 (Jak3), as well as Signal transducer and activator of transcription 5 (Stat5), are required for normal T cell function and survival. Constitutive activation of Jak3 and Stat5 have been linked to cancers of hematopoietic origin, including certain lymphomas and leukemias. The production of cAMP by adenylate cyclase has been shown to negatively regulate human TCR mediated cell proliferation. Since cAMP has been shown to negatively regulate T cell activation, we sought to investigate whether crosstalk exists between cAMP and IL-2R signaling. The first objective of this study was to determine the effect of cAMP on the activation of IL-2R signaling molecules Jak3 and Stat5. We found that the potent adenylate cyclase activator, forskolin, inhibited IL-2 activation of Jak3 and Stat5. Indeed, in vitro kinase assays and electrophoretic mobility shift assays verified a loss of Jak3 enzymatic activity and Stat5 DNA binding ability, respectively. Further analysis of IL-2R signaling showed that forskolin treatment reduced IL-2 induced association of the IL-2Rβ and γc chain. Because cAMP activates protein kinase A (PKA), the second objective was to determine the role for PKA in the cAMP directed regulation of IL-2R signaling intermediates. Interestingly, forskolin induced serine phosphorylation of Jak3, suggesting that cAMP can directly regulate Jak3 via activation of a serine/threonine kinase. Indeed, phosphoamino acid analysis revealed that PKA was able to induce Jak3 serine phosphorylation in the human leukemia cell line MT-2. In addition, in vitro kinase assays established that PKA can directly inhibit Jak3 enzymatic activity. Collectively, these data indicate that cAMP negatively regulates IL-2R signaling via various effector molecules by a previously unrecognized mechanism. This new data suggests that the Jak3/Stat5 pathway may be regulated by various pharmacological agents that stimulate cAMP production and thus can be used to uncouple some types of T cell mediated diseases

JAK1 Pseudokinase V666G Mutant Dominantly Impairs JAK3 Phosphorylation and IL-2 Signaling

Overactive Janus kinases (JAKs) are known to drive leukemia, making them well-suited targets for treatment. We sought to identify new JAK-activating mutations and instead found a JAK1-inactivating pseudokinase mutation, V666G. In contrast to other pseudokinase mutations that canonically lead to an active kinase, the JAK1 V666G mutation led to under-activation seen by reduced phosphorylation. To understand the functional role of JAK1 V666G in modifying kinase activity we investigated its influence on other JAK kinases and within the Interleukin-2 pathway. JAK1 V666G not only inhibited its own activity, but its presence could inhibit other JAK kinases. These findings provide new insights into the potential of JAK1 pseudokinase to modulate its own activity, as well as of other JAK kinases. Thus, the features of the JAK1 V666 region in modifying JAK kinases can be exploited to allosterically inhibit overactive JAKs

The Genomic Landscape of a Restricted ALL Cohort from Patients Residing on the U.S./Mexico Border

Next-generation sequencing (NGS) has identified unique biomarkers yielding new strategies in precision medicine for the treatment of Acute lymphoblastic leukemia (ALL). Hispanics show marked health disparities in ALL, often absent in clinical trials or cancer research. Thus, it is unknown whether Hispanics would benefit equally from curated data currently guiding precision oncology. Using whole-exome sequencing, nine ALL patients were screened for mutations within genes known to possess diagnostic, prognostic and therapeutic value. Genes mutated in Hispanic ALL patients from the borderland were mined for potentially pathogenic variants within clinically relevant genes. KRAS G12A was detected in this unique cohort and its frequency in Hispanics from the TARGET-ALL Phase II database was three-fold greater than that of non-Hispanics. STAT5B N642H was also detected with low frequency in Hispanic and non-Hispanic individuals within TARGET. Its detection within this small cohort may reflect a common event in this demographic. Such variants occurring in the MAPK and JAK/STAT pathways may be contributing to Hispanic health disparities in ALL. Notable variants in ROS1, WT1, and NOTCH2 were observed in the ALL borderland cohort, with NOTCH2 C19W occurring most frequently. Further investigations on the pathogenicity of these variants are needed to assess their relevance in ALL

Phosphorylation of Tyrosine 841 Plays a Significant Role in JAK3 Activation

Janus Kinase 3 (JAK3) plays a key role in the development, proliferation, and differentiation of various immune cells. It regulates gene expression by phosphorylation of Signal Transducers and Activators of Transcriptions (STATs) via the JAK/STAT pathway. Recently, we found a new JAK3 phosphorylation site, tyrosine 841 (Y841). The results showed that pY841 helps the kinase domain flip around the pseudo kinase domain, which may cause JAK3 conformational changes. It also reduces the size of the cleft between the N-lobe and the C-lobe of the JAK3 kinase domain. However, pY841 was found to enlarge the cleft when ATP/ADP was bound to the kinase. The increase in the cleft size suggested that pY841 enhanced the elasticity of the kinase domain. For unphosphorylated JAK3 (JAK3-Y841), the binding forces between the kinase domain and ATP or ADP were similar. After phosphorylation of Y841, JAK3-pY841 exhibited more salt bridges and hydrogen bonds between ATP and the kinase than between ADP and the kinase. Consequently, the electrostatic binding force between ATP and the kinase was higher than that between ADP and the kinase. The result was that compared to ADP, ATP was more attractive to JAK3 when Y841 was phosphorylated. Therefore, JAK3-pY841 tended to bind ATP rather than ADP. This work provides new insights into the role of phosphorylation in kinase activation and ATP hydrolysis and sheds light on the importance of understanding the molecular mechanisms that regulate the kinase function

Evaluation of CTLA-4 Blockage with Metronomic Chemotherapy for the Treatment of Preclinical Breast Cancer

The targeting of the CTLA-4 protein with the antibody ipilimumab has been a success in terms of producing an increase in the survival of patients with unresectable melanoma, and clinical trials are ongoing to evaluate this strategy in other tumor types. Our aim in this study was to evaluate the combination of CTLA-4 blocking with metronomic chemotherapy regimens. To that end, we subcutaneously implanted murine EMT-6 breast tumor cells into syngeneic Balb/c mice (n=6-8/group) and evaluated therapies on the established tumors. Murine CTLA-4 blocking was achieved using anti-mouse CD152 (CTLA-4), clone 9H10, injected on day 1 (100ug/mouse) and on day 6 (35ug/mouse) of therapy. Anti-CTLA-4 therapy was administered on its own or combined with metronomic regimens. These included; a) Bolus (150mg/kg, i.p.) cyclophosphamide (CTX) followed by metronomic CTX (20mg/kg/day, p.o.), b) metronomic CTX, and c) sequential gemcitabine therapy (160mg/kg every 3 days, i.p.) given to the tumors relapsing after the anti-CTLA-4 therapy. We observed that control (saline) treated tumors, or tumors treated with Bolus CTX plus metronomic CTX, grew rapidly and had to be sacrificed 4 weeks after tumor implantation. Anti-CTLA-4 monotherapy produced an initial tumor regression followed by tumor relapses, 2-3 weeks later, in 5/6 mice. Surprisingly, the Bolus CTX plus metronomic CTX hindered the effective CTLA-4 therapy, failed to produce tumor regression, and resulted in rapidly growing tumors. The combination of anti-CTLA-4 plus metronomic CTX also produced tumor regression and resulted in a longer delay in the appearance of relapsing tumors (p \u3c 0.05 compared to anti-CTLA-4 alone), which also eventually appeared in 5/6 mice. A Kaplan Meier plot showed that the anti-CTLA-4 plus metronomic CTX regimen significantly improved survival compared to the anti-CTLA-4 monotherapy (p \u3c 0.05). The regimen involving first line anti-CTLA4 therapy followed by a second line gemcitabine therapy, produced a sustained tumor regression that continued for over 100 days. In this group, 5/7 mice did not show a tumor regrowth; 1 mouse showed a tumor regrowth under continuous gemcitabine therapy with concomitant development of lung metastasis. Tumor cells lines were derived from the relapsing tumor and from the lung metastasis. Collectively our data shows that Bolus plus metronomic CTX may compromise anti-CTLA-4 therapy. Furthermore, anti-CTLA-4 therapy may be effectively combined with metronomic CTX, or with a sequential gemcitabine therapy, in a preclinical model of breast cancer