JAK Family Research Articles

The Novel SYK/JAK Inhibitor Cerdulatinib Demonstrates Good Tolerability and Clinical Response in a Phase 2a Study in Relapsed/Refractory Peripheral T-Cell Lymphoma and Cutaneous T-Cell Lymphoma

The Novel SYK/JAK Inhibitor Cerdulatinib Demonstrates Good Tolerability and Clinical Response in a Phase 2a Study in Relapsed/Refractory Peripheral T-Cell Lymphoma and Cutaneous T-Cell Lymphoma

Application of Sequential Palladium Catalysis for the Discovery of Janus Kinase Inhibitors in the Benzo[ c]pyrrolo[2,3- h][1,6]naphthyridin-5-one (BPN) Series.

The present account describes the discovery and development of a new benzo[ c]pyrrolo[2,3- h][1,6]naphthyridin-5-one (BPN) JAK inhibitory chemotype that has produced selective JAK inhibitors. Sequential palladium chemistry was optimized for the rapid access to a focused library of derivatives to explore the structure-activity relationships of the new scaffold. Several compounds from the series displayed potencies in the low nanomolar range against the four members of the JAK family with various selectivity profiles. Compound 20a, with an azetidine amide side chain, showed the best selectivity for JAK1 kinase vs JAK2, JAK3, and TYK2, with low nanomolar potency (IC50 = 3.4 nM). On the other hand, BPNs 17b and 18 had good general activity against the JAK family with excellent kinome selectivity profiles. Many of the new BPNs inhibited JAK3-mediated STAT-5 phosphorylation, the production of inflammatory cytokines, and the proliferation of primary T cells. Moreover, BPN 17b showed very similar in vivo results to tofacitinib in a rheumatoid arthritis animal model.

Janus kinase JAK1 maintains the ovarian reserve of primordial follicles in the mouse ovary

Is the Janus kinase and signal transducer and activator of transcription (JAK-STAT) signalling pathway involved in ovarian follicle development and primordial follicle activation? JAK1 is a key factor involved in the regulation of primordial follicle activation and maintenance of the ovarian reserve. A series of integrated, intrinsic signalling pathways (including PI3K/AKT, mTOR and KITL) are responsible for regulating the ovarian reserve of non-growing primordial follicles and ultimately female fertility. The JAK-STAT signal transduction pathway is highly conserved with established roles in cell division and differentiation. Key pathway members (specifically JAK1, STAT3 and SOCS4) have been previously implicated in early follicle development. A laboratory animal study was undertaken using the C57Bl/6 inbred mouse strain as a model for human ovarian follicle development. To determine which Jak genes were most abundantly expressed during primordial follicle activation, mRNA expression was analysed across a developmental time-course, with ovaries collected from female mice at post-natal days 1 (PND1), 4 (PND4), 8 (PND8), as well as at 6 weeks (6WK) and 7 months (7MTH) (n ≥ 4). Functional analysis of JAK1 was performed on PND2 mouse ovaries subjected to in vitro explant culture treated with 12.5 μM Ruxolitinib (JAK inhibitor) or vehicle control (DMSO) for 48 h prior to histological assessment (n ≥ 4). The expression and localization of the JAK family during ovarian follicle development in the C57Bl/6 inbred mouse strain were evaluated using quantitative PCR, immunoblotting and immunolocalisation. Functional studies were undertaken using the JAK inhibitor Ruxolitinib to investigate the underpinning cellular mechanisms via biochemical in vitro inhibition and histological assessment of intact neonate ovaries. All experiments were replicated at least three times using tissue from different mice unless otherwise stated. Jak1 is the predominant Jak mRNA expressed in the C57Bl/6 mouse ovary across all developmental time-points assessed (P ≤ 0.05). Forty-eight hour inhibition of JAK1 with Ruxolitinib of PND2 ovaries in vitro demonstrated concomitant acceleration of primordial follicle activation and apoptosis (P ≤ 0.001) and upregulation of downstream JAK-STAT pathway members STAT3 and suppressors of cytokine signalling 4 (SOCS4). N/A. Results are shown in one species, the C57Bl/6 mouse strain as an established model of human ovary development. Ruxolitinib also inhibits JAK2, with decreased efficacy. However, Jak2 mRNA had limited expression in the mouse ovary, particularly at the neonatal stages of follicle development, thus any effect of Ruxolitinib on primordial follicle activation was unlikely to be mediated via this isoform. This study supports a key role for JAK1 in the maintenance and activation of primordial follicles, with potential for targeting the JAK-STAT pathway as a method of regulating the ovarian reserve and female fertility. This project has been funded by the Australian National Health and Medical Research Council (G1600095) and The Hunter Medical Research Institute Bob and Terry Kennedy Children's Research Project Grant in Pregnancy & Reproduction (G1501433). All authors declare no conflict of interests.

In vitro and in vivo characterization of the JAK1 selectivity of upadacitinib (ABT-494)

BackgroundAnti-cytokine therapies such as adalimumab, tocilizumab, and the small molecule JAK inhibitor tofacitinib have proven that cytokines and their subsequent downstream signaling processes are important in the pathogenesis of rheumatoid arthritis. Tofacitinib, a pan-JAK inhibitor, is the first approved JAK inhibitor for the treatment of RA and has been shown to be effective in managing disease. However, in phase 2 dose-ranging studies tofacitinib was associated with dose-limiting tolerability and safety issues such as anemia. Upadacitinib (ABT-494) is a selective JAK1 inhibitor that was engineered to address the hypothesis that greater JAK1 selectivity over other JAK family members will translate into a more favorable benefit:risk profile. Upadacitinib selectively targets JAK1 dependent disease drivers such as IL-6 and IFNγ, while reducing effects on reticulocytes and natural killer (NK) cells, which potentially contributed to the tolerability issues of tofacitinib.MethodsStructure-based hypotheses were used to design the JAK1 selective inhibitor upadacitinib. JAK family selectivity was defined with in vitro assays including biochemical assessments, engineered cell lines, and cytokine stimulation. In vivo selectivity was defined by the efficacy of upadacitinib and tofacitinib in a rat adjuvant induced arthritis model, activity on reticulocyte deployment, and effect on circulating NK cells. The translation of the preclinical JAK1 selectivity was assessed in healthy volunteers using ex vivo stimulation with JAK-dependent cytokines.ResultsHere, we show the structural basis for the JAK1 selectivity of upadacitinib, along with the in vitro JAK family selectivity profile and subsequent in vivo physiological consequences. Upadacitinib is ~ 60 fold selective for JAK1 over JAK2, and > 100 fold selective over JAK3 in cellular assays. While both upadacitinib and tofacitinib demonstrated efficacy in a rat model of arthritis, the increased selectivity of upadacitinib for JAK1 resulted in a reduced effect on reticulocyte deployment and NK cell depletion relative to efficacy. Ex vivo pharmacodynamic data obtained from Phase I healthy volunteers confirmed the JAK1 selectivity of upadactinib in a clinical setting.ConclusionsThe data presented here highlight the JAK1 selectivity of upadacinitinib and supports its use as an effective therapy for the treatment of RA with the potential for an improved benefit:risk profile.

Dependency on the TYK2/STAT1/MCL1 axis in anaplastic large cell lymphoma

TYK2 is a member of the JAK family of tyrosine kinases that is involved in chromosomal translocation-induced fusion proteins found in anaplastic large cell lymphomas (ALCL) that lack rearrangements activating the anaplastic lymphoma kinase (ALK). Here we demonstrate that TYK2 is highly expressed in all cases of human ALCL, and that in a mouse model of NPM-ALK-induced lymphoma, genetic disruption of Tyk2 delays the onset of tumors and prolongs survival of the mice. Lymphomas in this model lacking Tyk2 have reduced STAT1 and STAT3 phosphorylation and reduced expression of Mcl1, a pro-survival member of the BCL2 family. These findings in mice are mirrored in human ALCL cell lines, in which TYK2 is activated by autocrine production of IL-10 and IL-22 and by interaction with specific receptors expressed by the cells. Activated TYK2 leads to STAT1 and STAT3 phosphorylation, activated expression of MCL1 and aberrant ALCL cell survival. Moreover, TYK2 inhibitors are able to induce apoptosis in ALCL cells, regardless of the presence or absence of an ALK-fusion. Thus, TYK2 is a dependency that is required for ALCL cell survival through activation of MCL1 expression. TYK2 represents an attractive drug target due to its essential enzymatic domain, and TYK2-specific inhibitors show promise as novel targeted inhibitors for ALCL.

"Do We Know Jack" About JAK? A Closer Look at JAK/STAT Signaling Pathway.

Janus tyrosine kinase (JAK) family of proteins have been identified as crucial proteins in signal transduction initiated by a wide range of membrane receptors. Among the proteins in this family JAK2 has been associated with important downstream proteins, including signal transducers and activators of transcription (STATs), which in turn regulate the expression of a variety of proteins involved in induction or prevention of apoptosis. Therefore, the JAK/STAT signaling axis plays a major role in the proliferation and survival of different cancer cells, and may even be involved in resistance mechanisms against molecularly targeted drugs. Despite extensive research focused on the protein structure and mechanisms of activation of JAKs, and signal transduction through these proteins, their importance in cancer initiation and progression seem to be underestimated. This manuscript is an attempt to highlight the role of JAK proteins in cancer biology, the most recent developments in targeting JAKs, and the central role they play in intracellular cross-talks with other signaling cascades.

Activation of the IL-6/JAK2/STAT3 pathway induces plasma cell mastitis in mice

Plasma cell mastitis (PCM) is a chronic mastitis with limited treatment options and common recurrence. A histopathological hallmark of PCM is the infiltration of numerous plasma cells surrounding the mammary duct. Our previous study showed that the activity of the IL-6/STAT3 signaling pathway was elevated in patients with PCM. However, the etiology of PCM remains largely unclear. In this study, we sought to explore the effects of IL-6/JAK2/STAT3 signaling pathway in the pathogenesis of PCM. Histological analysis showed that the mammary glands of mice that received human breast tissue homogenates, followed by an injection of IL-6, exhibited features of PCM similar to human PCM. The IL-6/JAK2/STAT3 signaling activity was significantly elevated and Bcl-2 was highly expressed in CD138 + plasma cells in the mammary glands of mice with PCM. Furthermore, treatment with AG-490, an inhibitor of JAK family kinases, suppressed activation of the IL-6/JAK2/STAT3 signaling cascade, in turn resulting in a decreased number of plasma cells in the mammary gland and reversing the pathogenesis of PCM. Taken together, our study indicated that a PCM mouse model was successfully established through activation of the IL-6/JAK2/STAT3 pathway by injecting IL-6 into the mammary gland of mouse that had received homogenates of human breast tissue. Thus, the IL-6/JAK2/STAT3 signaling pathway plays a critical role in orchestrating the pathogenesis of PCM.

Jak-TGFβ cross-talk links transient adipose tissue inflammation to beige adipogenesis.

The transient activation of inflammatory networks is required for adipose tissue remodeling including the "browning" of white fat in response to stimuli such as β3-adrenergic receptor activation. In this process, white adipose tissue acquires thermogenic characteristics through the recruitment of so-called beige adipocytes. We investigated the downstream signaling pathways impinging on adipocyte progenitors that promote de novo formation of adipocytes. We showed that the Jak family of kinases controlled TGFβ signaling in the adipose tissue microenvironment through Stat3 and thereby adipogenic commitment, a function that was required for beige adipocyte differentiation of murine and human progenitors. Jak/Stat3 inhibited TGFβ signaling to the transcription factors Srf and Smad3 by repressing local Tgfb3 and Tgfb1 expression before the core transcriptional adipogenic cascade was activated. This pathway cross-talk was triggered in stromal cells by ATGL-dependent adipocyte lipolysis and a transient wave of IL-6 family cytokines at the onset of adipose tissue remodeling induced by β3-adrenergic receptor stimulation. Our results provide insight into the activation of adipocyte progenitors and are relevant for the therapeutic targeting of adipose tissue inflammatory pathways.

Oncogenic activation of JAK3-STAT signaling confers clinical sensitivity to PRN371, a novel selective and potent JAK3 inhibitor, in natural killer/T-cell lymphoma

Aberrant activation of the JAK3-STAT signaling pathway is a characteristic feature of many hematological malignancies. In particular, hyperactivity of this cascade has been observed in natural killer/T-cell lymphoma (NKTL) cases. Although the first-in-class JAK3 inhibitor tofacitinib blocks JAK3 activity in NKTL both in vitro and in vivo, its clinical utilization in cancer therapy has been limited by the pan-JAK inhibition activity. To improve the therapeutic efficacy of JAK3 inhibition in NKTL, we have developed a highly selective and durable JAK3 inhibitor PRN371 that potently inhibits JAK3 activity over the other JAK family members JAK1, JAK2, and TYK2. PRN371 effectively suppresses NKTL cell proliferation and induces apoptosis through abrogation of the JAK3-STAT signaling. Moreover, the activity of PRN371 has a more durable inhibition on JAK3 compared to tofacitinib in vitro, leading to significant tumor growth inhibition in a NKTL xenograft model harboring JAK3 activating mutation. These findings provide a novel therapeutic approach for the treatment of NKTL.

Spectrum of somatic mutations and their prognostic significance in adult patients with B cell acute lymphoblastic leukemia

目的探讨成人急性B淋巴细胞白血病（B-ALL）患者的基因突变谱，并分析其对患者预后的影响。方法收集113例2009年6月至2015年9月收治的成人B-ALL患者DNA标本，采用靶向特异的二代测序技术，针对血液恶性疾病相关的112种基因进行突变分析，通过多个数据库筛选出可能致病的基因突变，描述其发生频谱，并通过Kaplan-Meier、Cox回归模型分析突变基因对患者总生存（OS）和无复发生存（RFS）的影响。结果113例患者中103例（92.0%）发生至少一种基因突变，29例（25.6%）患者发生≥3种基因突变。所有患者中突变率较高的基因有SF1、FAT1、MPL、PTPN11、NRAS等，Ph阳性B-ALL和Ph阴性B-ALL患者中基因突变特点不尽一致。进一步分析基因突变对患者预后的影响，发现在Ph阴性B-ALL中伴JAK-STAT信号通路相关基因突变的患者（如JAK1、JAK2突变）较该信号通路未受影响的患者预后差（OS：P值分别为0.011和0.001；RFS：P值分别为0.014和<0.001），而伴PTPN11突变的B-ALL患者较不伴PTPN11突变的患者有较好的OS及RFS，但差异无统计学意义（P值均>0.05）；在Ph阳性B-ALL患者中，表观遗传学修饰相关的信号通路异常的患者预后较差（OS：P=0.038；RFS：P=0.047）。结论基因突变在成人B-ALL中存在普遍性，发生频谱因亚型而异，涉及多种信号通路，JAK家族相关基因突变常提示患者预后较差，突变基因之间的共存现象也预示着成人B-ALL患者的遗传复杂性和不稳定性。

JAK and STAT members in channel catfish: Identification, phylogenetic analysis and expression profiling after Edwardsiella ictaluri infection

The Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway is one of the main pleiotropic cascades used to transmit information from extracellular receptors to the nucleus, which results in DNA transcription and expression of genes involved in immunity, proliferation, differentiation, migration, apoptosis, and cell survival. Members of JAK family and STAT family have been extensively studied in different mammalian species because of their important roles in innate and adaptive immune responses. However, they have not been systematically studied among teleost fish species. In this study, five JAK family members and eight STAT family members were identified and characterized from channel catfish. Phylogenetic analysis was conducted to properly annotate these genes. Syntenic analysis was also conducted to establish orthology, and confirm the results from phylogenetic analysis. Compared to mammals, more members of the JAK and STAT family were identified in channel catfish genome. Expression of JAK and STAT family members was detected in healthy catfish tissues, but was induced in gill, liver, and intestine after bacterial challenge. Notably, the significant upregulation of STAT1b gene in catfish liver, gill and intestine after Edwardsiella ictaluri infection supported the notion that high STAT1 expression are involved in defense against pathogens. Collectively, the increased expression of JAK and STAT members in tested tissues suggested their crucial function in defending the host against pathogen invasion.

Loss of function JAK1 mutations occur at high frequency in cancers with microsatellite instability and are suggestive of immune evasion.

Immune evasion is a well-recognized hallmark of cancer and recent studies with immunotherapy agents have suggested that tumors with increased numbers of neoantigens elicit greater immune responses. We hypothesized that the immune system presents a common selective pressure on high mutation burden tumors and therefore immune evasion mutations would be enriched in high mutation burden tumors. The JAK family of kinases is required for the signaling of a host of immune modulators in tumor, stromal, and immune cells. Therefore, we analyzed alterations in this family for the hypothesized signature of an immune evasion mutation. Here, we searched a database of 61,704 unique solid tumors for alterations in the JAK family kinases (JAK1/2/3, TYK2). We used The Cancer Genome Atlas and Cancer Cell Line Encyclopedia data to confirm and extend our findings by analyzing gene expression patterns. Recurrent frameshift mutations in JAK1 were associated with high mutation burden and microsatellite instability. These mutations occurred in multiple tumor types including endometrial, colorectal, stomach, and prostate carcinomas. Analyzing gene expression signatures in endometrial and stomach adenocarcinomas revealed that tumors with a JAK1 frameshift exhibited reduced expression of interferon response signatures and multiple anti-tumor immune signatures. Importantly, endometrial cancer cell lines exhibited similar gene expression changes that were expected to be tumor cell intrinsic (e.g. interferon response) but not those expected to be tumor cell extrinsic (e.g. NK cells). From these data, we derive two primary conclusions: 1) JAK1 frameshifts are loss of function alterations that represent a potential pan-cancer adaptation to immune responses against tumors with microsatellite instability; 2) The mechanism by which JAK1 loss of function contributes to tumor immune evasion is likely associated with loss of the JAK1-mediated interferon response.

CTLA4 Promotes Tyk2-STAT3–Dependent B-cell Oncogenicity

CTL-associated antigen 4 (CTLA4) is a well-established immune checkpoint for antitumor immune responses. The protumorigenic function of CTLA4 is believed to be limited to T-cell inhibition by countering the activity of the T-cell costimulating receptor CD28. However, as we demonstrate here, there are two additional roles for CTLA4 in cancer, including via CTLA4 overexpression in diverse B-cell lymphomas and in melanoma-associated B cells. CTLA4-CD86 ligation recruited and activated the JAK family member Tyk2, resulting in STAT3 activation and expression of genes critical for cancer immunosuppression and tumor growth and survival. CTLA4 activation resulted in lymphoma cell proliferation and tumor growth, whereas silencing or antibody-blockade of CTLA4 in B-cell lymphoma tumor cells in the absence of T cells inhibits tumor growth. This inhibition was accompanied by reduction of Tyk2/STAT3 activity, tumor cell proliferation, and induction of tumor cell apoptosis. The CTLA4-Tyk2-STAT3 signal pathway was also active in tumor-associated nonmalignant B cells in mouse models of melanoma and lymphoma. Overall, our results show how CTLA4-induced immune suppression occurs primarily via an intrinsic STAT3 pathway and that CTLA4 is critical for B-cell lymphoma proliferation and survival. Cancer Res; 77(18); 5118-28. ©2017 AACR.

Abstract 4046: Targeting jak/stat adaptive mechanism with jak1 inhibitor azd4205 reduces residual disease and prolongs benefit of osimertinib

Abstract EGFR kinase inhibitors have provided tremendous benefit for non-small cell lung cancer (NSCLC) patients with EGFR driver mutations. However, many patients fail to respond or have responses of limited duration. Accumulating evidence suggests that the JAK1/STAT3 axis plays a role in tumor escape, and that combined inhibition of EGFR and JAK1 may drive more complete and durable responses. Here we report AZD4205, a potent ATP-competitive inhibitor that inhibits JAK1 with a Ki of 2.8 nM and exhibits excellent selectivity vs. other JAK family kinases and across the kinome. Combination of AZD4205 (dosed at 12.5, 25 or 50 mg/kg BID, PO) with Osimertinib (doses ranging from 1 to 25 mg/kg QD, PO) led to enhanced in vivo anti-tumor activity in several NSCLC xenograft models driven by mutant EGFR: H1975 (L858R/T790M), PC-9 (del19), and HCC827 (del19). In the H1975 model, after 18 days of treatment beginning with 189mm3 established tumors, treatment with osimertinib at 2.5 mg/kg led to tumor growth inhibition of 92% relative to vehicle. Addition of AZD4205 at 12.5 and 50 mg/kg led to 30% and 69% tumor regression, respectively. AZD4205 dosed at 12.5 and 50 mg/kg BID resulted in pSTAT3 (Y705) knockdown of 72% and 91% at peak plasma concentration (2H) respectively, and 42% and 86% at 8H. In the PC-9 model, after 22 days of treatment beginning with 234mm3 established tumors, osimertinib at 1 mg/kg induced an average 41% regression, and increased to 74% with the addition of 50 mg/kg BID AZD4205. The volume of vehicle treated tumors increased by 3.7-fold over this treatment period. In the HCC827 model, beginning with 127mm3 established tumors, osimertinib at 25 mg/kg induced a complete response in 100% of mice after 11 days of treatment, but tumors regrew to the average starting volume ~24 days after stopping treatment. When 50 mg/kg of AZD4205 was added to the osimertinib treatment, regrowth was delayed by an additional ~7 days. Combination activity was also observed in the PDX (patient-derived xenograft) model LG1049 (del19 and T790M EGFR mutations). Beginning with 189mm3 established tumors, osimertinib at 25 mg/kg induced an average 39% regression after 28 days of treatment, which increased to 56% with the addition of 25 mg/kg AZD4205 on a 4 days on / 3 days off / week schedule. AZD4205 treatment led to 67% knockdown of pSTAT3 after 4H of treatment in this model. Our experiments establish AZD4205 as a potent and selective JAK1 inhibitor with desirable biophysical properties. While additional work is needed to build a patient enrichment strategy, the data generated supports targeting the adaptive JAK1/STAT3 axis with JAK1 inhibitor AZD4205 in clinic as a strategy to reduce residual disease and prolong the benefit of Osimertinib in lung cancer patients with EGFR activating mutations. Citation Format: Huawei (Ray) Chen, Geraldine Bebernitz, Kirsten Bell, Erica Anderson, Nanhua Deng, Jason Kettle, Paul Lyne, Richard Woessner. Targeting jak/stat adaptive mechanism with jak1 inhibitor azd4205 reduces residual disease and prolongs benefit of osimertinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4046. doi:10.1158/1538-7445.AM2017-4046

Targeting Coronaviral Replication and Cellular JAK2 Mediated Dominant NF-\u03baB Activation for Comprehensive and Ultimate Inhibition of Coronaviral Activity

Tylophorine-based compounds exert broad spectral, potent inhibition of coronaviruses. NF-κB activation is a common pro-inflammatory response of host cells to viral infection. The aims of this study were to (i) find an effective combination treatment for coronaviral infections through targeting of the virus per se and cellular NF-κB activity; and (ii) to study the underling mechanisms. We found that tylophorine-based compounds target the TGEV viral RNA and effectively inhibit TGEV replication. NF-κB inhibition also leads to anti-TGEV replication. NF-κB activation induced by TGEV infection was found to be associated with two convergent pathways, IKK-2_IκBα/p65 and JAK2 mediated p65 phosphorylation, in swine testicular cells. JAK2 inhibition either by CYT387 (a JAK family inhibitor) or by silencing JAK2-expression revealed a dominant JAK2 mediated p65 phosphorylation pathway for NF-κB activation and resulted in NF-κB inhibition, which overrode the IκBα regulation via the IKK-2. Finally, tylophorine-based compounds work cooperatively with CYT387 to impart comprehensive anti-TGEV activities. The combination treatment, wherein a tylophorine compound targets TGEV and a JAK2 inhibitor blocks the alternative dominant NF-κB activation mediated by JAK2, is more effective and comprehensive than either one alone and constitutes a feasible approach for the treatment of SARS-CoV or MERS-CoV.

Molecular modeling-driven approach for identification of Janus kinase 1 inhibitors through 3D-QSAR, docking and molecular dynamics simulations

Janus kinase 1 (JAK 1) belongs to the JAK family of intracellular nonreceptor tyrosine kinase. JAK-signal transducer and activator of transcription (JAK-STAT) pathway mediate signaling by cytokines, which control survival, proliferation and differentiation of a variety of cells. Three-dimensional quantitative structure activity relationship (3 D-QSAR), molecular docking and molecular dynamics (MD) methods was carried out on a dataset of Janus kinase 1(JAK 1) inhibitors. Ligands were constructed and docked into the active site of protein using GLIDE 5.6. Best docked poses were selected after analysis for further 3 D-QSAR analysis using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) methodology. Employing 60 molecules in the training set, 3 D-QSAR models were generate that showed good statistical reliability, which is clearly observed in terms of r2ncv and q2loo values. The predictive ability of these models was determined using a test set of 25 molecules that gave acceptable predictive correlation (r2Pred) values. The key amino acid residues were identified by means of molecular docking, and the stability and rationality of the derived molecular conformations were also validated by MD simulation. The good consonance between the docking results and CoMFA/CoMSIA contour maps provides helpful clues about the reasonable modification of molecules in order to design more efficient JAK 1 inhibitors. The developed models are expected to provide some directives for further synthesis of highly effective JAK 1 inhibitors.

Mechanisms and consequences of Jak-STAT signaling in the immune system.

Kinases of the Jak ('Janus kinase') family and transcription factors (TFs) of the STAT ('signal transducer and activator of transcription') family constitute a rapid membrane-to-nucleus signaling module that affects every aspect of the mammalian immune system. Research on this paradigmatic pathway has experienced breakneck growth in the quarter century since its discovery and has yielded a stream of basic and clinical insights that have profoundly influenced modern understanding of human health and disease, exemplified by the bench-to-bedside success of Jak inhibitors ('jakinibs') and pathway-targeting drugs. Here we review recent advances in Jak-STAT biology, focusing on immune cell function, disease etiology and therapeutic intervention, as well as broader principles of gene regulation and signal-dependent TFs.

Selective Downregulation of JAK2 and JAK3 by an ATP-Competitive pan-JAK Inhibitor.

PF-956980 has been used previously as a JAK3-selective chemical probe in numerous cell-based experiments. Here, we report that not only is PF-956980 a pan-JAK ATP-competitive inhibitor but it also causes selective reduction of endogenous JAK2 and JAK3 protein levels in human primary immune cells (in a time-dependent manner), leaving the other JAK family members (JAK1 and TYK2) unchanged. We found that PF-956980 selectively downregulated JAK2 and JAK3 mRNA, corresponding to changes observed at the protein level. This work highlights therapeutic opportunities for the development of pharmacological inhibitors that also modulate the expression of their cognate binding proteins.

Abstract PR09: A novel chimeric antigen receptor containing JAK-STAT signaling domains mediates superior antitumor effects

Abstract Adoptive transfer of anti-CD19 CAR-engineered T cells have shown impressive and often durable clinical responses in patients with refractory and relapsed pre-B-cell acute lymphoblastic leukemia (ALL). However, clinical efficacy of anti-CD19 CAR-T cell therapy for lymphoma and chronic lymphocytic leukemia as well as CAR-T cell therapies that target solid tumors is not yet satisfactory. Optimal T cell activation and proliferation requires multiple signals including T cell receptor engagement (signal 1), costimulation (signal 2), and cytokine engagement (signal 3). To the best of our knowledge, CAR genes currently tested in the clinic contain a CD3z domain (signal 1) and costimulatory domain(s) (signal 2), but not a domain that can transmit signal 3. Although forced expression of cytokine genes in CAR-T cells can improve their persistence and antitumor effects, constitutive cytokine expression poses a risk for autonomous T cell growth causing cancers. In this study, we have developed a novel CAR design, 28-IL2RB-z (YXXQ), which can activate the JAK-STAT pathway in an antigen-dependent manner, recapitulating cytokine exposure upon TCR engagement. We incorporated a minimal cytoplasmic domain of the IL-2 receptor β (IL2RB) between CD28 and CD3z, which contains a box1 motif for the binding of JAK family protein tyrosine kinases and the tyrosine residue at position 510, an essential site for STAT5 association. To promote STAT3 recruitment, we introduced a YXXQ motif to the C-terminus end of CD3z (28-IL2RB-z (YXXQ)). T cells transduced with the novel CD19 CAR gene induced phosphorylation of both STAT3 and STAT5 proteins only upon CD19 encounter, and exhibited increased expression of JAK-STAT target genes. Moreover, upon stimulation with CD19+ target cells, these T cells, compared to 2nd and 3rd generation CAR-T cells, demonstrated superior proliferative capacity with increased cellular division and reduced activation-induced cell death. Interestingly, CD8+ T cells engineered with this novel CAR maintained a stem cell-like memory (TSCM) phenotype and polyfunctional cytokine secretion capacity even after repeated antigenic stimulation. Treatment of 28-IL2RB-z (YXXQ) CAR-T cells with a STAT3 inhibitor largely abrogated their proliferative advantage and decreased the frequency of TSCM cells, suggesting that STAT3 signaling played a predominant role in the maintenance of the TSCM phenotype. All the 2nd generation, 3rd generation, and our new generation CAR-T cells similarly demonstrated potent in vitro cytotoxicity and IFN-γ secretion upon initial exposure to CD19+ cells. However, our novel CAR-T cells were superior to the other CAR-T cells in both cytolytic activity and cytokine secretion following multiple antigen exposures. NSG mice were intravenously injected with the human CD19+ leukemia cell line, NALM6, and treated with adoptive transfer of each of the 2nd, 3rd, or new generation CAR-transduced T cells. The 28-IL2RB-z (YXXQ) new generation CAR-T cells demonstrated superior persistence compared to other generation CAR-T cells, which resulted in significantly better overall survival. Long-term persisting 28-IL2RB-z (YXXQ) CAR-T cells from treated mice secreted more IFN-γ when restimulated ex vivo, suggesting the maintenance of a functional and less-exhausted phenotype in vivo. Furthermore, the 28-IL2RB-z (YXXQ) CAR-T cells also demonstrated better in vivo persistence and therapeutic effects than the 28-z and 28-BB-z CAR-T cells in NSG mice inoculated with primary human CD19+ pre-B-ALL cells. Importantly, the 28-IL2RB-z (YXXQ) CD19 CAR-T cells did not exhibit any proliferative responses when adoptively transferred into leukemia-free mice, affirming that JAK-STAT signals are activated only when exposed to the antigen. Taken together, these results suggest that our new generation CAR has the potential to demonstrate superior antitumor effects with minimal toxicities in the clinic. Clinical translation of this novel CAR is warranted. This abstract is also being presented as Poster A73. Citation Format: Yuki Kagoya, Shinya Tanaka, Marcus O. Butler, Mark Minden, Naoto Hirano. A novel chimeric antigen receptor containing JAK-STAT signaling domains mediates superior antitumor effects. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr PR09.

STAT3 activation in infection and infection-associated cancer

The Janus kinase/signal transducers and activators for transcription (JAK/STAT) pathway plays crucial roles in regulating apoptosis, proliferation, differentiation, and the inflammatory response. The JAK/STAT families are composed of four JAK family members and seven STAT family members. STAT3 plays a key role in inducing and maintaining a pro-carcinogenic inflammatory microenvironment. Recent evidence suggests that STAT3 regulates diverse biological functions in pathogenesis of diseases, such as infection and cancer. In the current review, we will summarize the research progress of STAT3 activation in infection and cancers. We highlight our recent study on the novel role of STAT3 in Salmonella infection-associated colon cancer. Infection with bacterial AvrA-expressing Salmonella activates the STAT3 pathway, which induces the β-catenin signals and enhances colonic tumorigenesis. STAT3 may be a promising target in developing prevention and treatment for infectious diseases and infection-associated cancers.