JAK Family Of Kinases Research Articles

TYK2: an emerging therapeutic target in rheumatic disease.

Tyrosine kinase 2 (TYK2) is a member of the JAK kinase family of intracellular signalling molecules. By participating in signalling pathways downstream of type I interferons, IL-12, IL-23 and IL-10, TYK2 elicits a distinct set of immune events to JAK1, JAK2 and JAK3. TYK2 polymorphisms have been associated with susceptibility to various rheumatic diseases including systemic lupus erythematosus and dermatomyositis. In vitro and animal studies substantiate these findings, highlighting a role for TYK2 in diseases currently managed by antagonists of cytokines that signal through TYK2. Various inhibitors of TYK2 have now been studied in human disease, and one of these inhibitors, deucravacitinib, has now been approved for the treatment of psoriasis. Phase II trials of deucravacitinib have also reported positive results in the treatment of psoriatic arthritis and systemic lupus erythematosus, with a preliminary safety profile that seems to differ from that of the JAK1, JAK2 and JAK3 inhibitors. Two other inhibitors of TYK2, brepocitinib and ropsacitinib, are also in earlier stages of clinical trials. Overall, TYK2 inhibitors hold promise for the treatment of a distinct spectrum of autoimmune diseases and could potentially have a safety profile that differs from other JAK inhibitors.

Ritlecitinib (Litfulo) for severe alopecia areata.

The FDA has approved ritlecitinib (Litfulo – Pfizer), an oral JAK and TEC kinase family inhibitor, for treatment of severe alopecia areata in patients ≥12 years old. Ritlecitinib is the second oral drug to be approved in the US for treatment of severe alopecia areata; baricitinib (Olumiant), a JAK inhibitor, is approved only for use in adults.

The NPM1-TYK2 Chimeric Fusion Promotes Activation of STAT Family Signaling, Skewing Towards Tfh Functional Subset Differentiation and Mature T-Cell Lymphomagenesis

Introduction Peripheral T cell lymphomas are heterogeneous neoplasms of post thymic T cells with distinct clinical, pathological and genetic characteristics. We previously identified the NPM1-TYK2 fusion in patients with CD30+ lymphoproliferative disorders, including 15% of primary cutaneous ALK− anaplastic large cell lymphoma. The oncogenicity of NPM1-TYK2 and its effect on T cell differentiation, the molecular mechanisms and signaling pathways through which it drives lymphomagenesis invivo remain unknown. Methods To investigate the effect of NPM1-TYK2 on oncogenesis and T cell differentiation, we established a conditional transgenic model (CD4-NPM1-TYK2). To investigate cooperativity with Trp53, CD4-NPM1-TYK2 mice were crossed with Trp53 floxed mice to generate CD4ΔTrp53-NPM1-TYK2 mice with T cell-specific concomitant heterozygous loss of Trp53. Mice (n = 15 per genotype) were monitored over time for tumor development. Hematopoietic organs were harvested and subjected to histolopathological and immunophenotypic analysis. Serum cytokine and immunoglobulin profiles were analyzed by Luminex multiplex immunoassays. The effects of NPM1-TYK2 on the physiological function of T cells were examined by growth curves and cell cycle analysis using anti-CD3/CD28 stimulated CD4+ T cells from CD4-NPM1-TYK2 mice in vitro. To evaluate the signaling sequences of NPM1-TYK2 expression, unbiased mass spectrometry (MS)-driven phosphoproteomics was performed. The pharmacological effect of Deucravacitinib, an allosteric TYK2 inhibitor, on NPM1-TYK2+ murine T cells and human T cell lymphoma-derived cells were examined. ResultsCD4-NPM1-TYK2 mice showed significant splenomegaly (n = 20; 104.8 mg vs 86.5 mg) and developed lymphoma with hepatomegaly due to atypical neoplastic infiltrates. Notably, CD4ΔTrp53-NPM1-TYK2 mice (n = 5; 15.4 months) exhibited an earlier onset of lymphoma compared to CD4-NPM1-TYK2 mice (n = 4; 19.8 months). Immunophenotypic characterization of CD4+ T cells from the spleen of CD4-NPM1-TYK2 mice (n = 7) revealed an increase in effector T cells (CD44+/CD62L−, 51.5% vs 39.4%) and a decrease in naïve T cells (CD44−/CD62L+, 32.9% vs 44.8%), suggesting NPM1-TYK2 promotes effector T cell differentiation in vivo. Among all T helper cell subtypes, increased Tfh cells was observed in CD4-NPM1-TYK2 mice (n = 8; ICOS+/CXCR5+/PD1+/BCL6+, 27.4% vs 15.5%), indicating NPM1-TYK2 mediates the commitment of Tfh cell skewing in vivo. Serum cytokine profile of CD4-NPM1-TYK2 mice also showed elevated Tfh-related cytokines (n = 4; IL-1β, IL-4, IL-6, IL-10, IL-12). Tfh cells are crucial for the formation of germinal center (GC) B cells and their differentiation into plasma cells. Elevated expansion of GC B cells (n = 5; CD95+/PNA+, 4.8% vs 3.8%) was also observed. Increased plasma cells (B220−/CD138+, 9.1% vs 3.8%) and elevated immunoglobulins (n = 6; IgA, IgG2b) in the serum corroborated observed lymphoplasmacytic cells in the histology of CD4-NPM1-TYK2 mice. Among MS-identified phosphoproteins demonstrating the YX(2-5)L motif of JAK family kinase substrates, we observed elevated levels of tyrosine phosphopeptides implicating STAT1/3/5 activation. Western blotting of CD4+ T cells from CD4-NPM1-TYK2 mice revealed hyperphosphorylation of STAT1/3/5. NPM1-TYK2+ T cells (n = 3 per genotype) were hyperreactive to stimulation in vitro, displaying increased activated cells (CD69+/CD25+, 83/80% vs 76/73%). The growth curves of NPM1-TYK2+ T cells showed increased growth (2.6-fold) with more proliferating cells in the S phase of cell cycle (31% vs 25%) whilst control CD4+ T cells stopped proliferating 10 days post-stimulation. Intriguingly, the NPM1-TYK2-mediated growth-promoting effect was attenuated by Deucravacitinib. Conclusion Conditional transgenic expression of NPM1-TYK2 in mature T-cell lineage promotes Tfh cell differentiation accompanied by intense GC B cell and plasma cell expansion, and hepatosplenomegaly with disseminated lymphoma accelerated by concomitant heterozygous ablation of Trp53in vivo. The biologic effects of NPM1-TYK2 expression are propagated through STAT1/3/5 activation. These data provide novel insights on the signaling, cellular immunologic and oncogenic consequences of NPM1-TYK2 expression in CD4+ T cells. Our studies implicate NPM1-TYK2 as a de facto oncogene in vivo and provides a valuable pre-clinical model for precision medicine. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

JAK1 inactivation promotes proliferation and migration of endometrial cancer cells via upregulating the hypoxia-inducible factor signaling pathway

BackgroundLoss-of-function (LOF) mutations of JAK1, a member of the JAK kinase family, were frequently observed in EC, indicating that JAK1 may act as a tumor suppressor, at least in EC. However, the mechanism of JAK1 mediated regulation of tumorigenesis remains poorly understood.MethodsThe genetic alterations of JAK1 in EC using latest sequencing dataset of EC deposited in TCGA database. The RNA-Seq dataset of EC and normal endometrial tissues from TCGA cohort was analyzed. The expression of JAK1 in EC and normal endometrial tissues were investigated using immunohistochemistry. The expression levels of genes in endometrial cancer cells were detected by quantitative reverse transcription-PCR (RT-qPCR) and western blotting. JAK1 protein was efficiently depleted by the two shRNAs. HIF1/2-α protein was efficiently depleted by siRNAs. JAK1 overexpressed EC cells were generated by an expressing plasmid. The proliferation and migration ability of cancer cells were evaluated by CCK8, colony formation assays and transwell assays. The global transcriptomic changes in JAK1-depleted KLE cells were investigated using RNA-Seq. Gene Ontology (GO) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were used to identify the most significant pathways that were altered in JAK1-depleted KLE cells. The physical association between HIF-1/2α and JAK1 using co-immunoprecipitation (co-IP) assays.ResultsIn the present study, we found that JAK1 was frequently mutated and downregulated in EC. JAK1 knockdown promotes EC cell proliferation and migration. JAK1 overexpression reduces EC cell proliferation and migration. We examined the transcriptional profiling changes in JAK1-depleted EC cells and unexpectedly found that the hypoxia inducible factor (HIF) pathway was activated. Mechanistically, JAK1 interacts with HIF-1/2α, and reduces HIF1/2-α protein expression under hypoxia. HIF-1/2α knockdown reverses the JAK1 knockdown–induced growth and migration of EC cells under hypoxia. JAK1 knockdown or pharmacological inhibition of JAK1 kinase activity by Ruxolitinib upregulates transcription of HIF target genes under hypoxia. JAK1 overexpression downregulates transcription of HIF target genes under hypoxia.ConclusionsThese findings provide novel insights into the functional link between JAK1 LOF mutations and abnormal HIF pathway activation in EC and suggest that pharmacological inhibition of HIF1/2 represents a promising therapeutic strategy targeting JAK1-mutated ECs.3W7_hN6trihWN8_decuxdBVideo

P991: IMPAIRMENT OF TCR SIGNALLING IN PATIENTS WITH MYELOFIBROSIS TREATED WITH RUXOLITINIB

Background: Ruxolitinib (rux) is a JAK1/JAK2 inhibitor with varied immune effects including on T cell, NK cell and dendritic cell function. Therapeutic effects are largely attributed to marked reduction in pro-inflammatory cytokines. Aims: We hypothesised that impairment of T cell receptor (TCR) signalling through off-target binding of Src kinases, including Lck, would be an additional mechanism of immune dysfunction. Methods: We performed phosphoflow cytometry in Tregs, T effectors and NK cells to assess the effect on signalling downstream from the TCR and activating NK cell receptors. 11-colour flow cytometry was performed after cells were activated with H2O2 for 15 minutes, due to its activity as a potent phosphatase inhibitor and cells were analysed for phosphorylation of ZAP70 as a surrogate of TCR signalling and STAT5. A gating strategy of CD4+/CD25+/FOXP3+/CD127lo cells was used for identification of Tregs. Results: Phosphoflow analysis was performed in 7 patients with a diagnosis of MF managed with rux and compared with 7 healthy controls (HC). 4 (57%) of patients were male and mean age was 61 (range 44-76). Patients were taking a median rux dosage of 30mg daily (10-50). Three patients had a DIPPS+ score of Int-2, 2 Int-1 and 2 low-risk. Results of phosphoflow analysis are expressed as relative fluorescence intensity (RFI) defined as MFI in H2O2 stimulated sample/MFI in unstimulated sample. As expected, patients on rux had significantly reduced phosphorylation of pSTAT5 when compared with HC in all cell subsets evaluated. The mean RFI of pSTAT5 in CD4+ and CD8+ cells in HC was 21.9 and 31.1 respectively, compared with 6 and 8.5 in rux patients (p=0.001/p=0.002). In CD56+ cells this was 46.2 vs 7.7 (p=0.005) and in Tregs this was 21.9 vs 6.1 (p=0.019). Of note, a difference in phosphorylation was also observed for pZAP70 indicative of inhibition of TCR proximal signalling. For pZAP70 mean RFI in CD4+ cells was 8.8 in controls compared with 2.2 in rux patients (p=0.05). In CD8+ cells mean RFI was 11.4 in controls and 2.4 in rux patients (p=0.04). In CD56+ cells mean RFI was 17.8 vs 2.4 (p=0.02), whilst in Tregs, significance was not reached with mean RFI of 6.3 vs 2.2 (p=0.13). Univariate analysis of rux patients showed no effect of age, comorbidities, DIPPS+ score or rux dosage on TCR or STAT5 signalling. Findings were compared with those observed in 4 patients with CML and deep molecular response on nilotinib, in view of the highly specific ABL1 binding properties of this TKI. As expected, patients on nilotinib had significantly higher RFI for pSTAT5 compared with rux patients, at 23, 32.8, 21.2 and 46.5 in CD4+, CD8+, Treg and NK cells respectively (p=0.002/p=0.002/p=0.006/p=0.003). Interestingly, patients on nilotinib also had significantly higher RFI for pZAP70 in CD4+, CD8+ and NK cells at 6.7, 7.4 and 8.1 compared with rux patients (p=0.035/p=0.025/p=0.037). Image:Summary/Conclusion: Studies have shown that rux inhibits T cell proliferation and reduces CD4+ cell cytokine secretion. Previous in vitro analysis of rux on CD4+ cells failed to identify an effect on TCR signalling after CD3/CD28 stimulation. In our ex-vivo analysis, using H2O2 stimulation, we observe TCR signalling inhibition in CD4+, CD8+ and NK cells, although reduced compared with effects on STAT5. It has been shown that rux inhibits Lck with an IC50 of 3.6uM. Whilst this is significantly less than the inhibitory effect on JAK kinases, there is ~34% sequency homology between Src and JAK family kinases in the kinase domain, suggesting off target kinase inhibition is implicated in the impairment of TCR signalling.

Characterization of ARTS-011, a potent and selective TYK2 inhibitor for the treatment of inflammatory disease

Abstract The JAK family kinase TYK2 is a key regulator of IL-12 and IL-23 signaling pathways, which have been shown to be involved in the pathogenesis of multiple inflammatory and autoimmune diseases such as psoriasis, lupus and inflammatory bowel diseases (IBD). TYK2 inhibition is an attractive therapeutic strategy for human autoimmune diseases. Here, we report a potent oral TYK2 allosteric inhibitor ARTS-011, with high selectivity and potency through optimized binding to the JH2 regulatory domain of the TYK2 protein. ARTS-011 potently binds to TYK2 JH2 domain in binding assay and inhibits IL-23 and IFNα/β induced reporter activation depending on TYK2 kinase. ARTS-011 also inhibits IFNα triggered pSTAT5 potently in human whole blood assay. In addition, ARTS-011 excellent selectivity against other JAK family kinases and panels of receptors, transporters, ion channels, CYP enzymes and the hERG channel. Furthermore, ARTS-011 was also investigated in multiple animal models including IL12/18-induced serum IFNγ mouse model, IL-23-induced mouse psoriasis model and CD40 antibody-induced mouse inflammatory bowel disease (IBD) model. In vivo pharmacodynamic study indicates that ARTS-011 significantly reverses IL12/18-induced IFNγ production in mouse. ARTS-011 also significantly reduces the ear swelling in a dose-dependent manner in IL-23-induced mouse psoriasis model. Lastly, ARTS-011 was highly efficacious in anti-CD40 antibody induced mouse IBD model. In summary, ARTS-011 is a highly selective, potent and orally available small molecule TYK2 inhibitor with suitable pharmaceutical properties as potential therapeutics in inflammatory disorders.

Tyrosine kinases in KMT2A/MLL-rearranged acute leukemias as potential therapeutic targets to overcome cancer drug resistance.

Aim: The main goal of this study was to elucidate at the transcript level the tyrosine kinase expression profiles of primary leukemia cells from mixed lineage leukemia 1 gene rearranged (KMT2A/MLL-R+) acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) patients. Methods: We evaluated protein tyrosine kinase (PTK) gene expression profiles of primary leukemic cells in KMT2A/MLL-R+ AML and ALL patients using publicly available archived datasets. Results: Our studies provided unprecedented evidence that the genetic signatures of KMT2A/MLL-R+ AML and ALL cells are characterized by transcript-level overexpression of specific PTK. In infants, children and adults with KMT2A/MLL-R+ ALL, as well as pediatric patients with KMT2A/MLL-R+ AML, the gene expression levels for FLT3, BTK, SYK, JAK2/JAK3, as well as several SRC family PTK were differentially amplified. In adults with KMT2A/MLL-R+ AML, the gene expression levels for SYK, JAK family kinase TYK2, and the SRC family kinases FGR and HCK were differentially amplified. Conclusion: These results provide new insights regarding the clinical potential of small molecule inhibitors of these PTK, many of which are already FDA/EMA-approved for other indications, as components of innovative multi-modality treatment platforms against KMT2A/MLL-R+ acute leukemias.

PHF6 and JAK3 Mutations Cooperatively Drive T-Cell Acute Lymphoblastic Leukemia Progression

T cell acute lymphoblastic leukemia (T-ALL) is one of the most frequent hematologic malignancies resulted from gene mutations and/or genomic rearrangements that occur in T cell progenitors. The 5-year survival rate of T-ALL patients is less than 50%. Much efforts have been dedicated to decipher the molecular events underlying TALL transformation, with the goals to identify specific therapeutic targets and develop new and more effective drugs. As a member of JAK kinase family, JAK3 mutations can be identified in 16.1% of T-ALL cases, and JAK3 M511I mutation is the most common one within all JAK3 mutations. Activating JAK3 M511I mutation induced a lympho-proliferative disorder, that followed by a T-ALL-like disease. PHF6 mutation is one of the most common co-existing gene mutations with JAK3 in T-ALL patients. Co-mutation events of JAK3 and PHF6 account for 1.89%-10.0% in T-ALL cases. However, the role(s) of PHF6 and JAK3 co-mutations in tumorigenesis is unknown.Here in this study, we first analyzed the genetic data of 449 T-ALL cases from multiple clinical centers in which JAK/STAT mutations is about 21% of the total cases. Interestingly, we found that PHF6 mutations were significantly associated with JAK3 mutations in these T-ALL cases (P<0.05), and the JAK3 and PHF6 co-mutation occurred in 7.2% of the cohort. Significantly, the average survival time of PHF6 and JAK/STAT co-mutated group was much shorter than that of the single JAK/STAT mutated group (P<0.05) or none-PHF6/JAK/STAT mutated group (P<0.001). We generated Phf6 KO+JAK3 M511I mice by transplanting JAK3M511I infected Phf6 Lin - cells into wild-type mice. All Phf6 KO+JAK3M511Imice succumbed to leukemia from 74 to 101 days after transplantation with significantly shorter survival time than that of Phf6WT+JAK3M511Imice. The Phf6 KO+JAK3M511I mice showed more aggressive phenotypes of T-ALL than Phf6WT+JAK3M511I mice, including higher counts of WBCs, neutrophils and lymphocytes in peripheral blood, as well as higher degree of extramedullary infiltration in spleen, liver, lung and brain. Extreme limiting dilution transplantation assays demonstrated a marked increase in leukemia-initiating cell activity in Phf6 KO +JAK3M511Icells when compared with Phf6WT +JAK3M511Icells, supporting a role for loss of Phf6 in promoting leukemia blast self-renewal and proliferation.To investigate the underlying molecular mechanisms of Phf6 in accelerating T-ALL development,we performed RNAseq to analyze the transcriptome programing changes associated with Phf6 in isogenic Phf6WT +JAK3M511I or Phf6KO +JAK3M511IT-ALL cells. Gene set enrichment analysis (GSEA) showed up-regulated cell cycle in Phf6 KO +JAK3 M511I leukemia cells. We then performed Chromatin immunoprecipitation sequencing (ChIP-seq) and found that PHF6 associated with BAI1 gene. qPCR and Western blot showed that the mRNA and protein expression of Bai1/BAI1 were significantly decreased in Phf6KO +JAK3M511Icells in comparison with Phf6 WT+JAK3M511I cells. BAI1 has been reported to prevent MDM2-mediated P53 ubiquitination, and loss of BAI1 reduces P53 level. We found that the protein expression of BAI1 and P53 was decreased, and the ubiquitination of P53 was significantly increased in PHF6 knockdown (KD) MOLT-4 cells (T-ALL) when compared with the control cells. BAI1 overexpression in PHF6 KD MOLT-4 cells significantly increased P53 expression in PHF6 KD+BAI1 OE MOLT-4 cells when compared with that of PHF6 KD cells. It suggested that Phf6 deficiency increased BAI1-mediated P53 degradation. To confirm this, we treated Phf6 KO + JAK3M511I mice with a JAK3 inhibitor (ruxolitinib) in combination with a MDM2 inhibitor (idasanutlin), and found a synergistic response of the treatment within a significant attenuation in leukemia burden in vivo.In conclusion, we found that PHF6 mutation frequently co-existed with JAK3 mutation in T-ALLs, and together they can drive an aggressive leukemia in mice model. Notably, PHF6 deficiency promoted JAK3-induced T-ALL progression by inhibiting BAI1-MDM2-P53 signaling pathway that is independent of JAK3-STAT signaling pathway. We further demonstrated that combination therapy by tofacitinib and idasanutlin can reduce the Phf6 KO and JAK3 M511I leukemia burden in vivo. Our study suggested that combinational usage of JAK3 inhibitors and MDM2 inhibitors may increase the drug benefit for T-ALL patients with PHF6 and JAK3 co-mutations. DisclosuresNo relevant conflicts of interest to declare.

Persistent Activation of JAK/STAT Signaling Plays an Important Role inin VitroJaki Resistance inTYK2-rearranged B-Cell Acute Lymphoblastic Leukaemia

Introduction TYK2-rearrangements have recently been detected in high-risk acute lymphoblastic leukemia (HR-ALL) cases and are associated with poor outcome. The resultant fusion protein is predominantly driven by Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling. Thus, JAK/TYK2 inhibitors (JAKi) are among the most promising targeted therapeutics against these fusions. This was confirmed in aMYB-TYK2mouse model where the induced aggressive B-ALL was effectively targeted by the novel dual SYK/JAKi, cerdulatinib (cerd) (Tavakoliet al.2020, EHA abstract EP353). Despite the clinical benefit of JAKi in myeloproliferative neoplasms, resistance occurs resulting in relapse. Hence it is necessary to identify potential JAKi-mediated resistance mechanisms inTYK2-rearranged B-ALL patients. This study modeled cerd resistance mechanisms to recapitulate possible clinical scenarios. Methods Ba/F3 pro-B cells were retrovirally transduced with a plasmid construct containing theMYB-TYK2fusion gene isolated from an ALL patient. A cerd resistant line (cerdres) was generated by exposure of Ba/F3-MYB-TYK2cells to increasing concentrations of cerd (up to 3µM; clinically achievable plasma level is 1-2µM) over a period of 151 d. IC50 was determined via CellTiter-Glo proliferation assay. Downstream signaling was determined by phospho-flow analysis. Sanger sequencing was performed over theMYB-TYK2fusion gene to identify emergence of mutations. Site directed mutagenesis of theMYB-TYK2fusion construct was used to model an identified mutationin vitro. Computational modeling of theTYK2mutation and cerd docking was performed via ICM-Pro (Molsoft L.C.C.). The effect of long-term exposure to cerd on activation of JAK family kinases was investigated via western blot. A cerd resensitized line (cerdresen) was generated by culturing cerdres Ba/F3-MYB-TYK2cells in cerd free media for 5 weeks. Results Long-term exposure of Ba/F3-MYB-TYK2cells to cerd resulted in resistance with an 8.7-fold increase in IC50 compared to vehicle control cells (DMSO exposed) (IC50=6508 vs 739nM,p=0.001; Figure 1A). A novel mutation in the kinase domain ofTYK2(p.R987Q, c.3338G&amp;gt;A) was identified in ~50% of cerdres Ba/F3-MYB-TYK2cells. However,de novointroduction ofMYB-TYK2p.R987Q into parental Ba/F3 cells indicated that resistance to cerd was not due to the mutation alone, as these cells displayed no significant decreased sensitivity to cerd compared with control cells (IC50=1200 vs 739nM,p&amp;gt;0.05; Figure A). Computational modeling indicated the binding orientation of cerd to the mutated kinase domain was reversed 180º resulting in less favourable binding (TYK2p.R987Q vsTYK2, binding score= -18.6 vs -23.4). Phosphoflow analysis demonstrated increased JAK/STAT signalling in cerdres Ba/F3-MYB-TYK2compared with control cells (MFI= 35.2 vs 16.5,p=0.008) that persisted despite TYK2 kinase inhibition (MFI= 29.8 vs 2.3,p=0.004; Figure B). Expression of p.R987Q mutation did not result in increased pSTAT5 levels in Ba/F3-MYB-TYK2p.R987Q. Given that JAK2 heterodimerisation with other JAK proteins can lead to JAK/STAT activation and drug persistence (Meyeret al.2017), other kinases may facilitate phosphorylation of TYK2 in cerdres Ba/F3-MYB-TYK2.Western blot analysis confirmed a significant increase in TYK2 phosphorylation (p=0.01) and JAK1 expression (p=0.0008) in cerdres vs control Ba/F3-MYB-TYK2cells (Figure C). Cerd withdrawal resulted in potential resensitization of cerdres Ba/F3-MYB-TYK2to cerd with an associated decrease in IC50 (cerdres vs cerdresen, 6508 vs 2603nM,p=0.0003). However, IC50 levels did not decrease to levels observed in control cells (cerdresen vs control, 2603 vs 739nM, p=0.01), potentially due to increased activation of STAT5 from cerd-induced accumulation of pTYK2 (Tvorogovet al. 2018). Conclusions In vitromodeling suggests that persistent JAK/STAT activation is due to changes in TYK2 expression. Possible heterodimer formation with JAK1 in the setting of JAKi -cerd- exposure allows cells to become resistant. Consequently, the novel evidence of resistance mechanisms to JAKi, provide a rationale for the use of other small molecule inhibitors (e.g. HSP90i and HDACi), to potentially retain TYK2 degradation ability in resistant cells. This targeted approach may contribute to the treatment of patient withTYK2-rearranged ALL. Disclosures White: Bristol-Myers Squibb:Honoraria, Research Funding;Amgen:Honoraria.

Molecular docking analysis of human JAK2 with compounds from tomatoes.

Janus kinase 2 (JAK2) is a tyrosine kinase receptor that belongs to the JAK family kinases is linked to oral cancer. We describe the molecular binding analysis of JAK2 with 23 compounds from tomotoes. Docking data shows five compounds (rutin, qucertin, narigenin, chlrogenia acid & kaempferol) with optimal binding features with JAK2 for further consideration.

Activation of STAT transcription factors by the Rho-family GTPases.

The Rho-family of small GTPases are biological molecular switches that are best known for their regulation of the actin cytoskeleton. Through their activation and stimulation of downstream effectors, the Rho-family control pathways involved in cellular morphology, which are commonly activated in cancer cell invasion and metastasis. While this makes them excellent potential therapeutic targets, a deeper understanding of the downstream signalling pathways they influence will be required for successful drug targeting. Signal transducers and activators of transcription (STATs) are a family of transcription factors that are hyper-activated in most cancer types and while STATs are widely understood to be activated by the JAK family of kinases, many additional activators have been discovered. A growing number of examples of Rho-family driven STAT activation, largely of the oncogenic family members, STAT3 and STAT5, are being identified. Cdc42, Rac1, RhoA, RhoC and RhoH have all been implicated in STAT activation, contributing to Rho GTPase-driven changes in cellular morphology that lead to cell proliferation, invasion and metastasis. This highlights the importance and therapeutic potential of the Rho-family as regulators of non-canonical activation of STAT signalling.

Abstract 5313: TT-00420, a novel multiple kinase inhibitor to treat TNBC

Abstract TT-00420, a clinical stage spectrum-selective multiple kinase inhibitor, has been shown to be very efficacious against TNBC cell lines and in vivo models in our previous study. TT-00420 simultaneously inhibits Aurora A/B, RTKs involved in angiogenesis, JAK kinase family as well as CSF1R in kinase profiling assay. In breast cancer cell panel screening, TT-00420 specifically inhibits the proliferation across different subtypes of TNBC, while leaving ER/PR+ and Her2+ cells intact. Incubation of TNBC cell line HCC1806 with TT-00420 results in dose-dependent down-regulation of EGFR, TWIST1, SNAI2, FOSL1, and MYC, a cluster of genes collectively known as “Achilles cluster” essential for TNBC cell growth and proliferation. TT-00420 exhibits great in vivo efficacy in a panel of TNBC PDX models. In the mechanism study, we reveal that the inhibition of Aurora A or B lays the cornerstone of pan-TNBC inhibition capability of TT-00420, and the inhibition of angiogenesis pathway or JAK family or EGFR contributes to potency and activity of TT-00420 against TNBC at various degrees. Treatment of a mouse syngeneic TNBC model with TT-00420 normalizes the vessel conditions in tumors, which is consistent with TT-00420's capability to enhance the anti-tumor immune response both alone or combined with PD-1 blockade. Taken together, TT-00420 represents a novel synergistic inhibitory mechanism to treat TNBC. Citation Format: Peng Peng, Guoyu Li, Xiaoyan Qiang, Yuwei Tian, Qi Yu, Elisabetta Marangoni, Frank Wu. TT-00420, a novel multiple kinase inhibitor to treat TNBC [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5313.

Discovery of Tyrosine Kinase 2 (TYK2) Inhibitor (PF-06826647) for the Treatment of Autoimmune Diseases.

Tyrosine kinase 2 (TYK2) is a member of the JAK kinase family that regulates signal transduction downstream of receptors for the IL-23/IL-12 pathways and type I interferon family, where it pairs with JAK2 or JAK1, respectively. On the basis of human genetic and emerging clinical data, a selective TYK2 inhibitor provides an opportunity to treat autoimmune diseases delivering a potentially differentiated clinical profile compared to currently approved JAK inhibitors. The discovery of an ATP-competitive pyrazolopyrazinyl series of TYK2 inhibitors was accomplished through computational and structurally enabled design starting from a known kinase hinge binding motif. With understanding of PK/PD relationships, a target profile balancing TYK2 potency and selectivity over off-target JAK2 was established. Lead optimization involved modulating potency, selectivity, and ADME properties which led to the identification of the clinical candidate PF-06826647 (22).

17 AZD4205, A SELECTIVE, GI TRACT-ENRICHED SELECTIVE JAK1 INHIBITOR FOR CROHN’S DISEASE: PRECLINICAL EVIDENCE AND PHASE I DATA

Abstract Background AZD4205 is an oral, ATP-competitive, JAK1 selective inhibitor. Nonclinical data showed its higher drug concentration within the gastrointestinal (GI) tract relative to plasma in the rodents, suggesting its potential as an effective and safe treatment option for patients with Crohn’s disease (CD). AZD4205 also has been evaluated in a phase I study in healthy volunteers (HVs). Here we report AZD4205 preclinical data in cells and animal models, and phase I data from HVs. Method Enzymatic activity of AZD4205 was assessed in in vitro biochemical assay with human isolated JAK kinase domains. The cellular activity of AZD4205 was evaluated in human peripheral blood mononuclear cells (PBMCs) by assessing cytokine induced phosphorylated-STATs (pSTATs). The in vivo efficacy of AZD4205 was assessed in a murine CD model induced by subcutaneous injection of 10 mg/kg indomethacin, and AZD4205 was orally administered at different doses for 5 consecutive days. The ileum and bone marrow tissues were collected post AZD4205 treatment to measure pSTAT3 expression using immunohistochemistry. The drug concentration of AZD4205 in ileum and plasma were measured by HPLC. Phase I study of HVs (Clinicaltrial.gov Identifier: NCT03728023) comprised of single ascending dose (SAD) of 5 to 150 mg, food effect (FE) with 50 mg single dosing with or without food, and multiple ascending dose (MAD) of 25 to 100 mg once daily for 14 days. Safety monitoring was conducted, and blood samples were collected to assess PK of AZD4205. Result In enzymatic assay, AZD4205 exhibits greater than 200-400-fold selectivity over other JAK family kinases. AZD4205 inhibits pSTAT1, pSTAT3, and pSTAT5 in human PBMCs with IC50 of 50, 308, and 90 nM, respectively. The drug concentration of AZD4205 in murine ileum was around 100-fold higher than that in plasma. In murine CD model, the pSTAT3 was inhibited by 93% in ileum but only 43% in bone marrow at 4 hr post AZD4205 treatment. AZD4205 exhibited a dose-dependent effect on improving body weight loss and decreasing colon density in this model. In the NCT03728023 study, 66HVs were dosed with AZD4205 or matched placebo, SAD (n=20 in AZD4205, n=10 in placebo), FE (n=12 in AZD4205) and MAD (n=18 in AZD4205, n=6 in placebo). AZD4205 was well-tolerated, and no ≥ G3 drug related AE was observed. A dose-proportional increase of AZD4205 plasma concentrations was detected in HVs. The T1/2 was close to 48 hr and hence accumulation of about 3-fold in Cmax and AUC was observed after multiple once daily dosing. No relevant food effect was observed to significantly impact AZD4205 PK. Conclusion Based on preclinical murine data, AZD4205 has unique GI-tract enriched PK properties, as a promising agent for CD. Clinical data in HVs demonstrated its favorable safety and PK profiles. A phase II study in moderate to severe CD is planned.

SAT-091 Metabolic Effects of JAK1/2 Inhibition in Patients with Myeloproliferative Neoplasms

Background: Ruxolitinib is a JAK1/2 inhibitor that is FDA approved to treat certain myeloproliferative neoplasms (MPNs), including myelofibrosis (MF) and polycythemia vera (PV). The JAK family kinases are targeted in cancers due to their role in cytokine receptor signal transduction, and are also key intermediates in growth hormone, leptin and prolactin receptor signaling. Clinical trials have reported that ruxolitinib treatment was associated with weight gain, but other metabolic consequences remain unknown. Aim: We aimed to determine the metabolic consequences of JAK1/2 inhibition with ruxolitinib in patients with MPN in the clinical setting. Methods: IRB approval was obtained, and we identified patients from an electronic medical record (EMR) based database who began treatment with ruxolitinib for MPN from December 2011 to December 2016. Data collected from EMRs included age, gender, weight, height, systolic and diastolic blood pressure (SBP, DBP), medications, co-morbidities, random serum glucose, and lipid profile. We compared parameters from baseline (prior to initiating ruxolitinib) to 72±8 weeks after starting treatment. Baseline and 72 week data were compared using the Wilcoxon signed rank test. Results: 129 patients were initially identified, of which 71 had data available for weight, and at least one other metabolic parameter of interest at baseline and 72 weeks. Patient characteristics were as follows: 49.3% male; mean age at initiation of treatment was 64.6±SD11.1 years; indications for treatment were MF (77%), PV(17%), other MPNs (6%). Mean baseline weight was 73.6±16.9kg, and was 78.3±8.9kg at 72 weeks (n=71, p<0.001). Mean body mass index (BMI) at baseline was 25.6±4.6kg/m2, and 27.3±5.4kg/m2 at 72 weeks (n=68, p<0.001).The BMI (kg/m2) distribution for underweight (<18.5), normal (18.5-24.9), overweight (25.0-29.9), and obese (>30) at baseline were: 3%, 43%, 41%, and 13%, respectively; at 72 weeks it was 1%, 40%, 34%, and 28%, respectively (p=0.002). At 72 weeks,19% moved up a BMI class from baseline. Next, we investigated the effect of ruxolitinib treatment on BP and glucose concentrations. SBP was 124±15 mmHg at baseline and 129±18 mmHg at 72 weeks, (p=0.042, n=71). DBP was not different between baseline and 72 weeks. Glucose at baseline was 99±28mg/dL and 101±29mg/dL at 72 weeks (p=0.015, n=59). There was no change in the percent of patients with hyperglycemia (glucose ≥200mg/dL) or on treatment for diabetes at baseline (13.6%) and 72 weeks (13.6%). An insufficient number of patients had lipid data available for analysis. Conclusions: Systemic JAK1/2 inhibition was associated with weight gain, the development of obesity, and increased SBP in this cohort of patients. As pharmacological JAK1 and 2 inhibitors are developed and more widely used, it is important to gain a greater understanding of their long-term metabolic consequences.

Discovery of a JAK1/3 Inhibitor and Use of a Prodrug To Demonstrate Efficacy in a Model of Rheumatoid Arthritis.

The four members of the Janus family of nonreceptor tyrosine kinases play a significant role in immune function. The JAK family kinase inhibitor, tofacitinib 1, has been approved in the United States for use in rheumatoid arthritis (RA) patients. A number of JAK inhibitors with a variety of JAK family selectivity profiles are currently in clinical trials. Our goal was to identify inhibitors that were functionally selective for JAK1 and JAK3. Compound 22 was prepared with the desired functional selectivity profile, but it suffered from poor absorption related to physical properties. Use of the phosphate prodrug 32 enabled progression to a murine collagen induced arthritis (CIA) model. The demonstration of a robust efficacy in the CIA model suggests that use of phosphate prodrugs may resolve issues with progressing this chemotype for the treatment of autoimmune diseases such as RA.

Discovery of an Orally Available Janus Kinase 3 Selective Covalent Inhibitor.

JAK family kinases are important mediators of immune cell signaling and Janus Kinase 3 (JAK3) has long been indicated as a potential target for autoimmune disorders. Intensive efforts to develop highly selective JAK3 inhibitors have been underway for many years. However, because of JAK3's strong binding preference to adenosine 5'-triphosphate (ATP), a number of inhibitors exhibit large gaps between enzymatic and cellular potency, which hampers efforts to dissect the roles of JAK3 in cellular settings. Using a targeted covalent inhibitor approach, we discovered compound 32, which overcame ATP competition (1 mM) in the enzymatic assay, and demonstrated significantly improved inhibitory activity for JAK3-dependent signaling in mouse CTLL-2 and human peripheral blood mononuclear cells. Compound 32 also exhibited high selectivity within the JAK family and good pharmacokinetic properties. Thus, it may serve as a highly valuable tool molecule to study the overlapping roles of JAK family kinases in complex biological settings. Our study also suggested that for covalent kinase inhibitors, especially those targeting kinases with low Km ATP values, the reversible interactions between molecules and proteins should be carefully optimized to improve the overall potency.

Checkpoint Inhibition in CSF3R Mutated Chronic Neutrophilic Leukemia

Mutations in the colony-stimulating factor 3 receptor (CSF3R) have been identified in 90% of chronic neutrophilic leukemia (CNL) and lower frequency in atypical chronic myeloid leukemia (aCML). It was shown recently, that signaling of the different types of CSF3R mutations varies, but a central role of the JAK/STAT pathway activation by oncogenic CSF3R membrane proximal point mutations has been anticipated. However, the exact downstream signaling pathways mediated by CSF3R mutations are still not completely understood. Recent studies in JAK mutated myeloproliferative diseases uncovered the vital importance of JAK/STAT signaling in PD-L1 mediated immune escape, thereby suggesting a possible role and mechanistic link for PD-1/PD-L1 immune checkpoint regulation in CNL and aCML.To investigate the dependence of the oncogenic CSF3R signaling cascade on an activated JAK/STAT axis, we performed Ruxolitinib treatment in MTT assays with BaF3 cells retrovirally infected with pMIG-R1 vectors harboring either CSF3R point or truncation mutations. Our data indicate, that both, CSF3R point and truncation mutations induced cell growth could be inhibited by Ruxolitinib treatment. To narrow down the involvement of the different JAK family kinases, we included CSF3R mutation transduced JAK1 deficient U4C, JAK2 deficient y2A and TYK2 deficient U1A cell lines in combination with different JAK inhibitors. Interestingly, here we were able to show that CSF3R point mutations require functional JAK1/TYK2-STAT3 signaling for oncogenic potential, whereas truncation mutations depend on all JAK kinases through STAT3/5 for activation.To further study potential therapeutic strategies, we examined checkpoint inhibition in CSF3R mutated disease. In this regard we demonstrate a significant induction of PD-L1 expression in murine cell lines upon expression of CSF3R point mutations T615A or T618I. To investigate the impact of PD-L1 upregulation in vivo, we used a retroviral murine bone marrow (BM) transplantation model where we infected BM of Balb/c mice with a vector carrying CSF3R T618I point mutation and transplanted it into lethally irradiated recipients. Transplanted mice developed a CSF3R driven leukemia and died by leukocytosis and organ infiltration, whereas mice transplanted with BM harboring CSF3RT618I and additional ADA mutation failed to induce leukemia. To test checkpoint inhibition as a potential therapeutic strategy in vivo, CSF3RT618I transplanted mice were treated regularly with anti-PD-L1-antibody or isotype control. Interestingly, checkpoint inhibition led to reduced EGFP burden and prolonged survival of responder mice suggesting a treatment response of immunooncologic agents in these rare diseases.In immunophenotypic analyses of primary patient material, we were able to show increased PD-L1 expression in primary diseased myeloid cells isolated from peripheral blood of CNL and aCML patients, thereby defining this structure as a possible new therapeutic target.To summarize, our data expand the knowledge of oncogenic CSF3R signaling pathways and implicate an immunotherapeutic strategy by checkpoint inhibition in CSF3R driven CNL and atypical CML. DisclosuresLubbert:Teva: Other: Study drug; Celgene: Other: Travel Grant; Janssen: Honoraria, Research Funding.

STAT3 Is Activated By DYRK1A and Is a Potential Therapeutic Target in B-ALL

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that mediates signal transduction from the extracellular surface to the nucleus. Canonically, STAT3 is phosphorylated at Tyrosine 705 (Y705) by JAK family kinases, which promotes its dimerization and subsequent localization to the nucleus. However, the role of Serine 727 (S727) phosphorylation in regulating STAT3 activity varies across cell types and remains unclear in hematopoietic tissues particularly. Several studies indicate that phosphorylation at S727 is critical for optimal STAT3 function. For example, astrogliogenesis is regulated by enhancing STAT3 activity by phosphorylation of S727 by DYRK1A. Of note, DYRK1A is overexpressed in Down syndrome-acute lymphoblastic leukemia (DS-ALL), and has previously been found to phosphorylate substrates in order to prime them for downstream phosphorylation events. Given these findings, we hypothesized that the DYRK1A phosphorylation of STAT3 at S727 is critical for promoting DS-ALL. Furthermore, certain subtypes of ALL have high rates of JAK2 activation, namely DS-ALL and Philadelphia-like ALL (Ph-like ALL); we propose that STAT3 can effectively be targeted specifically in these subtypes.In order to elucidate the role of DYRK1A phosphorylation of STAT3, we treated cytokine-deprived murine pre-B cells with EHT1610, a selective DYRK1 inhibitor, or vehicle and then pulsed the cells with JAK-STAT activating cytokines. EHT1610-treated cells had diminished S727 phosphorylation compared to vehicle, regardless of cytokine pulse; however, only vehicle-treated cells regained Y705 phosphorylation after cytokine pulse. This suggests that S727 phosphorylation is cytokine-independent and is critical for maintenance of Y705 phosphorylation. We then generated flag-tagged STAT3 S727 phospho-mimetic (S727D/E) and phospho-deficient (S727A) alleles and transduced them into pre-B cells. We observed that the degree of Y705 phosphorylation is dependent on S727, as cells expressing S727A have reduced Y705 phosphorylation compared to wild-type STAT3. Additionally, overexpression of the phospho-deficient allele conferred a significant proliferative impairment compared to the phospho-mimetic alleles.As DS-ALL and Ph-like ALL often have JAK2-activating mutations, we next aimed to determine if loss of S727 phosphorylation would decrease ALL cell growth. Indeed, two human Ph-like ALL cell lines, MHH-CALL4 and MUTZ5, displayed decreased proliferation when overexpressing the S727A mutant. These cell lines were also sensitive to treatment with C188-9, a small molecule STAT3 inhibitor that is in clinical trials for various solid tumors. Additionally, we treated primary patient ALL samples with amplification of HSA21 segments ex vivo and found that DS-ALL samples were preferentially sensitive to STAT3 inhibition compared to HD-ALL or iAMP-ALL, suggesting that STAT3 is specifically a target in JAK2-activated ALL.Our study provides new and significant insights into the regulation of STAT3 by DYRK1A, and presents a new therapeutic target for ALL cells with JAK2 activating mutations. DisclosuresBourquin:Amgen: Other: Travel Support. Crispino:Scholar Rock: Research Funding; Forma Therapeutics: Research Funding.

Tyrosine phosphorylation of the transmembrane protein SIRPα: Sensing synaptic activity and regulating ectodomain cleavage for synapse maturation

Synapse maturation is a neural activity-dependent process during brain development, in which active synapses preferentially undergo maturation to establish efficient neural circuits in the brain. Defects in this process are implicated in various neuropsychiatric disorders. We have previously reported that a postsynaptic transmembrane protein, signal regulatory protein-α (SIRPα), plays an important role in activity-dependently directing synapse maturation. In the presence of synaptic activity, the ectodomain of SIRPα is cleaved and released and then acts as a retrograde signal to induce presynaptic maturation. However, how SIRPα detects synaptic activity to promote its ectodomain cleavage and synapse maturation is unknown. Here, we show that activity-dependent tyrosine phosphorylation of SIRPα is critical for SIRPα cleavage and synapse maturation. We found that during synapse maturation and in response to neural activity, SIRPα is highly phosphorylated on its tyrosine residues in the hippocampus, a structure critical for learning and memory. Tyrosine phosphorylation of SIRPα was necessary for SIRPα cleavage and presynaptic maturation, as indicated by the fact that a phosphorylation-deficient SIRPα variant underwent much less cleavage and could not drive presynaptic maturation. However, SIRPα phosphorylation did not affect its synaptic localization. Finally, we show that inhibitors of the Src and JAK kinase family suppress neural activity-dependent SIRPα phosphorylation and cleavage. Together, our results indicate that SIRPα phosphorylation serves as a mechanism for detecting synaptic activity and linking it to the ectodomain cleavage of SIRPα, which in turn drives synapse maturation in an activity-dependent manner.