Hit the spleen, JAK!
Lane Steven W,Mullally Ann
Blood
In this issue of Blood, Wang et al report on the response of splenic-derived hematopoietic stem and progenitor cells from patients with myelofibrosis (MF) to the Janus kinase (JAK) inhibitor, AZD1480.
10.1182/blood-2014-09-600635
Inhibitor repurposing reveals ALK, LTK, FGFR, RET and TRK kinases as the targets of AZD1480.
Gudernova Iva,Balek Lukas,Varecha Miroslav,Kucerova Jana Fialova,Kunova Bosakova Michaela,Fafilek Bohumil,Palusova Veronika,Uldrijan Stjepan,Trantirek Lukas,Krejci Pavel
Oncotarget
Many tyrosine kinase inhibitors (TKIs) have failed to reach human use due to insufficient activity in clinical trials. However, the failed TKIs may still benefit patients if their other kinase targets are identified by providing treatment focused on syndromes driven by these kinases. Here, we searched for novel targets of AZD1480, an inhibitor of JAK2 kinase that recently failed phase two cancer clinical trials due to a lack of activity. Twenty seven human receptor tyrosine kinases (RTKs) and 153 of their disease-associated mutants were in-cell profiled for activity in the presence of AZD1480 using a newly developed RTK plasmid library. We demonstrate that AZD1480 inhibits ALK, LTK, FGFR1-3, RET and TRKA-C kinases and uncover a physical basis of this specificity. The RTK activity profiling described here facilitates inhibitor repurposing by enabling rapid and efficient identification of novel TKI targets in cells.
10.18632/oncotarget.22674
Structural Insights into JAK2 Inhibition by Ruxolitinib, Fedratinib, and Derivatives Thereof.
Journal of medicinal chemistry
The discovery that aberrant activity of Janus kinase 2 (JAK2) is a driver of myeloproliferative neoplasms (MPNs) has led to significant efforts to develop small molecule inhibitors for this patient population. Ruxolitinib and fedratinib have been approved for use in MPN patients, while baricitinib, an achiral analogue of ruxolitinib, has been approved for rheumatoid arthritis. However, structural information on the interaction of these therapeutics with JAK2 remains unknown. Here, we describe a new methodology for the large-scale production of JAK2 from mammalian cells, which enabled us to determine the first crystal structures of JAK2 bound to these drugs and derivatives thereof. Along with biochemical and cellular data, the results provide a comprehensive view of the shape complementarity required for chiral and achiral inhibitors to achieve highest activity, which may facilitate the development of more effective JAK2 inhibitors as therapeutics.
10.1021/acs.jmedchem.0c01952
JAK1-dependent transphosphorylation of JAK2 limits the antifibrotic effects of selective JAK2 inhibitors on long-term treatment.
Zhang Yun,Liang Ruifang,Chen Chih-Wei,Mallano Tatjana,Dees Clara,Distler Alfiya,Reich Adam,Bergmann Christina,Ramming Andreas,Gelse Kolja,Mielenz Dirk,Distler Oliver,Schett Georg,Distler Jörg H W
Annals of the rheumatic diseases
OBJECTIVES:Janus kinase 2 (JAK2) has recently been described as a novel downstream mediator of the pro-fibrotic effects of transforming growth factor-β. Although JAK2 inhibitors are in clinical use for myelodysplastic syndromes, patients often rapidly develop resistance. Tumour cells can escape the therapeutic effects of selective JAK2 inhibitors by mutation-independent transactivation of JAK2 by JAK1. Here, we used selective JAK2 inhibition as a model to test the hypothesis that chronic treatment may provoke resistance by facilitating non-physiological signalling pathways in fibroblasts. METHODS:The antifibrotic effects of long-term treatment with selective JAK2 inhibitors and reactivation of JAK2 signalling by JAK1-dependent transphosphorylation was analysed in cultured fibroblasts and experimental dermal and pulmonary fibrosis. Combined JAK1/JAK2 inhibition and co-treatment with an HSP90 inhibitor were evaluated as strategies to overcome resistance. RESULTS:The antifibrotic effects of selective JAK2 inhibitors on fibroblasts decreased with prolonged treatment as JAK2 signalling was reactivated by JAK1-dependent transphosphorylation of JAK2. This reactivation could be prevented by HSP90 inhibition, which destabilised JAK2 protein, or with combined JAK1/JAK2 inhibitors. Treatment with combined JAK1/JAK2 inhibitors or with JAK2 inhibitors in combination with HSP90 inhibitors was more effective than monotherapy with JAK2 inhibitors in bleomycin-induced pulmonary fibrosis and in adTBR-induced dermal fibrosis. CONCLUSION:Fibroblasts can develop resistance to chronic treatment with JAK2 inhibitors by induction of non-physiological JAK1-dependent transactivation of JAK2 and that inhibition of this compensatory signalling pathway, for example, by co-inhibition of JAK1 or HSP90 is important to maintain the antifibrotic effects of JAK2 inhibition with long-term treatment.
10.1136/annrheumdis-2016-210911
A structure-function perspective of Jak2 mutations and implications for alternate drug design strategies: the road not taken.
Gnanasambandan K,Sayeski P P
Current medicinal chemistry
Jak2 is a non-receptor tyrosine kinase that is involved in the control of cellular growth and proliferation. Due to its significant role in hematopoiesis, Jak2 is a frequent target for mutations in cancer, especially myeloid leukemia, lymphoid leukemia and the myeloproliferative neoplasms (MPN). These mutations are common amongst different populations all over the world and there is a great deal of effort to develop therapeutic drugs for the affected patients. Jak2 mutations, whether they are point, deletion, or gene fusion, most commonly result in constitutive kinase activation. Here, we explore the structure-function relation of various Jak2 mutations identified in cancer and understand how they disrupt Jak2 regulation. Current Jak2 inhibitors target the highly conserved active site in the kinase domain and therefore, these inhibitors may lack specificity. Based on our knowledge regarding structure-function correlations as they pertain to regulation of Jak2 kinase activity, an alternative approach for specific Jak2 targeting could be via allosteric inhibitor design. Successful reports of allosteric inhibitors developed against other kinases provide precedent for the development of Jak2 allosteric inhibitors. Here, we suggest plausible target sites in the Jak2 structure for allosteric inhibition. Such targets include the type II inhibitor pocket and substrate binding site in the kinase domain, the kinase-pseudokinase domain interface, SH2-JH2 linker region and the FERM domain. Thus, future Jak2 inhibitors that target these sites via allosteric mechanisms may provide alternative therapeutic strategies to existing ATP competitive inhibitors.
10.2174/092986711797379267
Dual PI3K/AKT/mTOR inhibitor BEZ235 synergistically enhances the activity of JAK2 inhibitor against cultured and primary human myeloproliferative neoplasm cells.
Molecular cancer therapeutics
Hemopoietic progenitor cells (HPC) from myeloproliferative neoplasms (MPN) such as myelofibrosis commonly express mutant JAK2-V617F or other mutations that are associated with increased activities of JAK-STAT5/3, RAS/RAF/MAPK, and PI3K/AKT/mTOR pathways. This confers proliferative and survival advantage on the MPN HPCs. Treatment with JAK tyrosine kinase inhibitor (TKI), for example, TG101209, TG101348 (SAR302503), or INCB018424 (ruxolitinib), inhibits mutant JAK2-mediated signaling. Although effective in reducing constitutional symptoms and splenomegaly, treatment with JAK-TKI does not ameliorate myelofibrosis or significantly improve survival of patients with advanced myelofibrosis. Here, we show that treatment with the dual phosphoinositide-3-kinase (PI3K)/AKT and mTOR inhibitor BEZ235 attenuated PI3K/AKT and mTOR signaling, as well as induced cell-cycle growth arrest and apoptosis of the cultured human JAK2-V617F-expressing HEL92.1.7 (HEL), UKE1 cells, and primary CD34+ myelofibrosis (MF)-MPN cells. Treatment with BEZ235 also induced significant apoptosis of the JAK2-TKI resistant HEL/TGR cells that were selected for resistance against JAK-TKI. Cotreatment with BEZ235 and JAK2-TKI (TG101209 and SAR302503) synergistically induced lethal activity against the cultured and primary CD34+ MPN cells while relatively sparing the normal CD34+ HPCs. These findings create a compelling rationale to determine the in vivo activity of dual PI3K/mTOR inhibitors in combination with JAK inhibitors against myelofibrosis HPCs.
10.1158/1535-7163.MCT-12-0862
Differences in gene expression and alterations in cell cycle of acute myeloid leukemia cell lines after treatment with JAK inhibitors.
European journal of pharmacology
Janus kinase (JAK) inhibitors are a promising treatment strategy in several hematological malignancies and autoimmune diseases. A number of inhibitors are in clinical development, and two have already reached the market. Unfortunately, all of them are burdened with different toxicity profiles. To check if the JAK inhibitors of different selectivity evoke different responses on JAK2-dependent and independent cells, we have used three acute myeloid leukemia cell lines with confirmed JAK2 mutation status. We have found that JAK inhibitors exert distinct effect on the expression of BCLXL, CCND1 and c-MYC genes, regulated by JAK pathway, in JAK2 wild type cells in comparison to JAK2 V617F-positive cell lines. Moreover, cell cycle analysis showed that inhibitors alter the cycle by arresting cells in different phases. Our results suggest that observed effect of JAK2 inhibitors on transcription and cell cycle level in different cell lines are associated not with activity within JAK family, but presumably with other off-target activities.
10.1016/j.ejphar.2015.08.037
JAK kinase targeting in hematologic malignancies: a sinuous pathway from identification of genetic alterations towards clinical indications.
Springuel Lorraine,Renauld Jean-Christophe,Knoops Laurent
Haematologica
Constitutive JAK-STAT pathway activation occurs in most myeloproliferative neoplasms as well as in a significant proportion of other hematologic malignancies, and is frequently a marker of poor prognosis. The underlying molecular alterations are heterogeneous as they include activating mutations in distinct components (cytokine receptor, JAK, STAT), overexpression (cytokine receptor, JAK) or rare JAK2 fusion proteins. In some cases, concomitant loss of negative regulators contributes to pathogenesis by further boosting the activation of the cascade. Exploiting the signaling bottleneck provided by the limited number of JAK kinases is an attractive therapeutic strategy for hematologic neoplasms driven by constitutive JAK-STAT pathway activation. However, given the conserved nature of the kinase domain among family members and the interrelated roles of JAK kinases in many physiological processes, including hematopoiesis and immunity, broad usage of JAK inhibitors in hematology is challenged by their narrow therapeutic window. Novel therapies are, therefore, needed. The development of more selective inhibitors is a questionable strategy as such inhibitors might abrogate the beneficial contribution of alleviating the cancer-related pro-inflammatory microenvironment and raise selective pressure to a threshold that allows the emergence of malignant subclones harboring drug-resistant mutations. In contrast, synergistic combinations of JAK inhibitors with drugs targeting cascades that work in concert with JAK-STAT pathway appear to be promising therapeutic alternatives to JAK inhibitors as monotherapies.
10.3324/haematol.2015.132142
Molecular pathways: molecular basis for sensitivity and resistance to JAK kinase inhibitors.
Meyer Sara C,Levine Ross L
Clinical cancer research : an official journal of the American Association for Cancer Research
Janus-activated kinases (JAK) are the mediators of a variety of cytokine signals via their cognate receptors that result in activation of intracellular signaling pathways. Alterations in JAK1, JAK2, JAK3, and TYK2 signaling contribute to different disease states, and dysregulated JAK-STAT signaling is associated with hematologic malignancies, autoimmune disorders, and immune-deficient conditions. Genetic alterations of JAK2 occur in the majority of patients with myeloproliferative neoplasms and occur in a subset of patients with acute leukemias. JAK-mediated signaling critically relies on STAT transcription factors, and on activation of the MAPK and PI3K/Akt signaling axes. Hyperactive JAK at the apex of these potent oncogenic signaling pathways therefore represents an important target for small-molecule kinase inhibitors in different disease states. The JAK1/2 inhibitor ruxolitinib and the JAK3 inhibitor tofacitinib were recently approved for the treatment of myelofibrosis and rheumatoid arthritis, respectively, and additional ATP-competitive JAK inhibitors are in clinical development. Although these agents show clinical activity, the ability of these JAK inhibitors to induce clinical/molecular remissions in hematologic malignancies seems limited and resistance upon chronic drug exposure is seen. Alternative modes of targeting JAK2 such as allosteric kinase inhibition or HSP90 inhibition are under evaluation, as is the use of histone deacetylase inhibitors. Combination therapy approaches integrating inhibition of STAT, PI3K/Akt, and MAPK pathways with JAK kinase inhibitors might be critical to overcome malignancies characterized by dysregulated JAK signaling.
10.1158/1078-0432.CCR-13-0279
Constitutive activation of JAK2 in mammary epithelium elevates Stat5 signalling, promotes alveologenesis and resistance to cell death, and contributes to tumourigenesis.
Caffarel M M,Zaragoza R,Pensa S,Li J,Green A R,Watson C J
Cell death and differentiation
Signalling through the janus kinase (JAK)/signal transducer and activator of transcription (Stat) pathway is required at different stages of mammary gland development, and this pathway is frequently hyper-activated in cancer, including tumours of the breast. Stats 3, 5 and 6 have important roles in the differentiation and survival of mammary alveolar cells, but somewhat paradoxically, both Stat3 and 5 can have oncogenic activity in the mammary gland. Constitutive activation of JAK2 could be anticipated to result in hyper-activation of Stats 1, 3, 5 and 6 with concomitant cell transformation, although the outcome is difficult to envisage, particularly since Stats 3 and 5 play opposing roles in normal mammary gland development. Here, we show that expression of a constitutively active JAK2 mutant, JAK2 V617F, leads to hyper-activation of Stat5 in mammary epithelial cells (MECs), and transgenic mice expressing JAK2 V617F specifically in the mammary gland exhibit accelerated alveologenesis during pregnancy and delayed post-lactational regression. Overexpressing JAK2 V617F in MECs in vitro results in elevated proliferation and resistance to cell death. Furthermore, constitutively active JAK2 enhances anchorage-independent cell growth in the presence of a co-operating oncogene and accelerates tumourigenesis in a xenograft model. Taken together, our results provide insights into signalling downstream of constitutively active JAK2 and could be important for understanding the molecular mechanisms of breast tumourigenesis.
10.1038/cdd.2011.122
A Novel Selective JAK2 Inhibitor Identified Using Pharmacological Interactions.
Lin Tony Eight,HuangFu Wei-Chun,Chao Min-Wu,Sung Tzu-Ying,Chang Chao-Di,Chen Yi-Ying,Hsieh Jui-Hua,Tu Huang-Ju,Huang Han-Li,Pan Shiow-Lin,Hsu Kai-Cheng
Frontiers in pharmacology
The JAK2/STAT signaling pathway mediates cytokine receptor signals that are involved in cell growth, survival and homeostasis. JAK2 is a member of the Janus kinase (JAK) family and aberrant JAK2/STAT is involved with various diseases, making the pathway a therapeutic target. The similarity between the ATP binding site of protein kinases has made development of specific inhibitors difficult. Current JAK2 inhibitors are not selective and produce unwanted side effects. It is thought that increasing selectivity of kinase inhibitors may reduce the side effects seen with current treatment options. Thus, there is a great need for a selective JAK inhibitor. In this study, we identified a JAK2 specific inhibitor. We first identified key pharmacological interactions in the JAK2 binding site by analyzing known JAK2 inhibitors. Then, we performed structure-based virtual screening and filtered compounds based on their pharmacological interactions and identified compound NSC13626 as a potential JAK2 inhibitor. Results of enzymatic assays revealed that against a panel of kinases, compound NSC13626 is a JAK2 inhibitor and has high selectivity toward the JAK2 and JAK3 isozymes. Our cellular assays revealed that compound NSC13626 inhibits colorectal cancer cell (CRC) growth by downregulating phosphorylation of STAT3 and arresting the cell cycle in the S phase. Thus, we believe that compound NSC13626 has potential to be further optimized as a selective JAK2 drug.
10.3389/fphar.2018.01379