JAK Kinase Inhibitors Research Articles

Synergism Between HOXA9 and Mutant JAK3 (M511I) Leads to Rapid Leukemia Development within an in Vivo Murine Bone Marrow Transplant Model

Activation mutations in JAK3 occur in 16% of T-cell acute lymphoblastic leukemia (T-ALL) cases, and co-occur frequently with HOXA cluster rearrangement. Genomic rearrangement of the HOXA cluster results in increased expression of HOXA9 and HOXA10. However it remains unclear if either HOXA9 or HOXA10 can cooperate with activating JAK3 mutations during oncogenic transformation and leukemogenesis. We have previously shown that JAK3 mutations lead to cell transformation and cause a long latency T-ALL in vivo using a mouse bone marrow transplant model. In this study we demonstrate that co-expression of the activating JAK3(M511I) protein with HOXA9 cooperate to develop leukemia within 30 days of transplant using an in vivo bone marrow transplant model. In our cooperative model, murine hematopoietic stem / progenitor cells were co-transduced with either both retroviral vectors encoding JAK3(M511I)/GFP and HOXA9/mCherry or each individually and then injected into sub-lethally irradiated recipient mice. Mice transplanted with bone marrow cells expressing JAK3(M511I) mutant alone developed T-ALL in 120 to 150 days. In sharp contrast, mice transplanted with cells expressing both JAK3(M511I) and HOXA9 showed rapid leukemia development within 30 days after transplant. Leukemia development was characterized by the rapid and specific increase in GFP-mCherry double positive cells. These animals showed high WBC, and splenomegaly and accumulation of immature CD8 single positive cells in the thymus. Similar experiments with HOXA10 did not show cooperation suggesting that HOXA9 is the more important oncogene in HOXA rearranged leukemias when a JAK3 activating mutation is present. To determine the underlying genetic mechanism for cooperation between HOXA9 and JAK3(M511I) the single positive JAK3 and double positive JAK3/HOXA9 expressing cells were isolated from thymi of leukemic mice for both epigenomic profiling using ATAC-seq and gene expression profiling. These analyses identified genetic pathways activated by the co-expression of HOXA9 and JAK3(M511I) mutation and provide mechanistic insight into the synergistic interaction between these two factors in driving leukemia development. Treatment of the animals with a JAK kinase inhibitor resulted in delayed leukemia development, confirming that the leukemia cells remain sensitive to the JAK inhibitor. This mouse model provides insight in the cooperation between oncogenes in leukemia development and provides a model for the study of targeted agents in this setting. DisclosuresNo relevant conflicts of interest to declare.

Comprehensive Kinase Profile of Pacritinib, a Non-Myelosuppressive JAK2 Kinase Inhibitor in Phase 3 Development in Primary and Post ET/PV Myelofibrosis

BackgroundPacritinib, a JAK2, JAK2 V617F and FLT3 kinase inhibitor is in Phase III development in myelofibrosis. In preclinical models and in Phase 1 studies in both non-Hodgkin lymphoma and myelofibrosis, it appeared unique among this class of agents in its lack of myelosuppression at doses that inhibited the JAK2/STAT3 pathway and resulted in significant clinical efficacy. This potentially important clinical observation was verified in two Phase II studies in myelofibrosis that had no exclusion restriction for low platelets. Pacritinib was effective at reducing spleen size and symptoms regardless of starting platelet counts and there was no apparent decline in either platelet count or hemoglobin levels, or an increase in transfusion requirements.Aims and Methods: To help elucidate the mechanisms for pacritinib’s lack of myelosuppression, a kinome screening analysis against 429 recombinant kinases at 100 nM pacritinib concentration was performed, followed by titration from 1-100nM against those that were >50% inhibited at 100nM (Anastassiadis et al.; Nature Biotechnology; 2011). In humans, pacritinib achieves steady state free drug levels of approximately 200nM at the standard 400 mg daily dose.Results:All kinases with IC50 values of <50nM are included in the table.TableKinaseIC50 (nM)JAK2; JAK2(V617F)6.0; 9.4FLT3; FLT3-ITD; FLT3 (D835Y)6.4; 12.0; 8.3TYK227.0JAK318.3IRAK1 (IL-1 receptor kinase)13.6HIPK4 (PT kinase for P53 ser-9)14.5FMS39.5c-KIT and most common mutations except (V560G), (V559D/V654A)43.8c-Src; (c-Src (T341M))47.0 (55.5)Of note, pacritinib did not affect JAK1 (82% control at 100nM). As the concentrations of pacritinib achieved clinically are likely inhibit signaling the kinases listed in the table, these data suggest that the lack of myelosuppression with pacritinib may be either due to inhibition of JAK2 without affecting JAK1 or to suppression of IRAK1, an IL-1 receptor kinase, downstream from Toll-Like Receptors, and associated with the inflammatory response and suppression of normal hematopoiesis in myelodysplasia models (Rhyasen et al.; Cancer Cell; 24:90, 2013).Conclusions: The kinase profile for pacritinib is unique among agents in development for myelofibrosis combining lack of suppression of JAK1 with suppression of inflammatory signaling through IRAK1. Moreover, the kinase inhibition profile of pacritinib suggests its potential therapeutic utility in AML, MDS, and particularly in CMML due to CMML’s upregulation of JAK2, c-FMS, and IRAK1. DisclosuresSinger:CTI BioPharma, Corp: Employment, Equity Ownership. Mesa:Incyte Corporation, CTI, NS Pharma, Inc., Gilead Science Inc., Celgene: Research Funding. Verstovsek:CTI BioPharma, Corp: Research Funding.

Abstract 2760: CSF3R ligand-independent mutations found in chronic neutrophilic leukemia have altered O-linked glycosylation

Abstract We have recently discovered that a large percentage (59%) of patients with chronic neutrophilic leukemia (CNL) or atypical chronic myeloid leukemia (aCML) have mutations in Colony Stimulating Factor 3 Receptor (CSF3R, aka GCSFR) (Maxson et al, NEJM 2013). These mutations are either point mutations in the extracellular domain (T618I, T615A) or truncations of the cytoplasmic domain. The most frequent mutation is T618I (aka T595I). The two classes of CSF3R mutations operate through distinct mechanisms that result in differential sensitivity to inhibition of tyrosine kinases downstream of CSF3R. While the mechanism of action of CSF3R truncation mutations has been well characterized-they have increased cell surface expression due to loss of negative regulatory motifs-the way in which the CSF3R point mutations activate the receptor is unclear. Consistent with a previous report, the CSF3R T618I mutation is ligand independent in both mouse bone marrow colony assays and the Ba/F3 pro-B cell line. WT CSF3R consists of two molecular weight species suggestive of differential posttranslational modification. Analysis of CSF3R point mutations T618I and T615A revealed an alteration of banding pattern of these mutations. Since extracellular threonine residues are capable of being O-glycosylated, we employed endoglycosidase digestion to reveal that the larger CSF3R species is O-glycosylated. Furthermore the T618I and T615A mutations reduce O-glycosylation of the receptor. This analysis was confirmed by direct labeling of O-glycosylation using azide-modified galactosamine, which labeled WT CSF3R, but had minimal labeling of CSF3R T618I. Receptor dimerization studies showed increased dimerization of the CSF3R T618I mutant relative to WT. Sequencing of CSF3R from an atypical CML patient samples revealed an additional mutation not previously observed in this disease, T640N. The T640N mutation (aka T617N) is a ligand-independent mutation originally found in a family with hereditary chronic neutrophilia (Plo et al, JEM 2009). Plo et al found that this mutation resides within the transmembrane domain and predicted by molecular modeling that it increased receptor dimerization through interaction of the transmembrane helices. In mouse bone marrow colony assays we confirmed the ligand-independence of the mutation and also found that it had similar potency as T618I in colony and cytokine-independent growth assays. Furthermore, like T618I, the T640N mutation conferred sensitivity to a JAK kinase inhibitor in colony assays. Surprisingly, the T640N mutation had an alteration of banding pattern very similar to T618I, suggesting that it may also have altered glycosylation status, even though the mutation resides within the transmembrane domain. Our studies demonstrate that O-glycosylation is critical for regulation of CSF3R dimerization and that the T618I and T640N mutations can activate the receptor to promote oncogenesis. Citation Format: Julia Maxson, Samuel Luty, Jason MacManiman, Samantha Savage, Melissa Abel, Swaleh Bahamadi, Brian Druker, Jeffrey Tyner. CSF3R ligand-independent mutations found in chronic neutrophilic leukemia have altered O-linked glycosylation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2760. doi:10.1158/1538-7445.AM2014-2760

C-Abl Activates Janus Kinase 2 in Normal Hematopoietic Cells

Jak2 is involved in cytokine growth factor-stimulated signal transduction, but the mechanism of its activation is largely unknown. Here, we investigated Jak2 activation in a normal hematopoietic cell line, 32D mouse myeloid cells. The bimolecular fluorescence complementation studies showed that c-Abl formed a stable complex with Jak2 in live cells. Co-immunoprecipitation results showed that c-Abl bound to the βc chain of IL-3/IL-5/GM-CSF receptors. The kinase activities of both c-Abl and Jak2 were stimulated by IL-3 in 32D cells. Decreasing c-Abl protein expression in 32D cells by inducible shRNA decreased Jak2 activity and resulted in the failure of Jak2 activation in response to IL-3. Treatment of IL-3 and serum-starved 32D cells with 1 μM imatinib mysylate inhibited IL-3 stimulated kinase activities of both c-Abl and Jak2. In addition, the kinase-deficient Bcr-Abl mutant (p210K1172R) was defective for activation of Jak2 in 32D cells and impaired IL-3 independent growth, which was rescued by overexpression of c-Abl (+Abl). IL-3 efficiently inhibited apoptosis of 32Dp210K/R+Abl cells induced by imatinib mysylate but not Jak2 kinase inhibitor TG101209. In summary, our findings provide evidence that the kinase function of c-Abl and its C-terminal CT4 region is crucial for its interaction with Jak2 and its activation. c-Abl kinase activity induced by IL-3 is required for IL-3-stimulated Jak2 and Jak1 activation. Our findings reveal a novel regulatory role of c-Abl in Jak2 activation induced by IL-3 cytokine growth factor in 32D hematopoietic cells.

High-throughput chemical screening identifies AG-490 as a stimulator of aquaporin 2 membrane expression and urine concentration.

A reduction or loss of plasma membrane aquaporin 2 (AQP2) in kidney principal cells due to defective vasopressin (VP) signaling through the VP receptor causes excessive urine production, i.e., diabetes insipidus. The amount of AQP2 on the plasma membrane is regulated by a balance of exocytosis and endocytosis and is the rate limiting step for water reabsorption in the collecting duct. We describe here a systematic approach using high-throughput screening (HTS) followed by in vitro and in vivo assays to discover novel compounds that enhance vasopressin-independent AQP2 membrane expression. We performed initial chemical library screening with a high-throughput exocytosis fluorescence assay using LLC-PK1 cells expressing soluble secreted yellow fluorescent protein and AQP2. Thirty-six candidate exocytosis enhancers were identified. These compounds were then rescreened in AQP2-expressing cells to determine their ability to increase AQP2 membrane accumulation. Effective drugs were then applied to kidney slices in vitro. Three compounds, AG-490, β-lapachone, and HA14-1 increased AQP2 membrane accumulation in LLC-PK1 cells, and both AG-490 and β-lapachone were also effective in MDCK cells and principal cells in rat kidney slices. Finally, one compound, AG-490 (an EGF receptor and JAK-2 kinase inhibitor), decreased urine volume and increased urine osmolality significantly in the first 2-4 h after a single injection into VP-deficient Brattleboro rats. In conclusion, we have developed a systematic procedure for identifying new compounds that modulate AQP2 trafficking using initial HTS followed by in vitro assays in cells and kidney slices, and concluding with in vivo testing in an animal model.

Synthesis and structure–activity relationships of 4-fluorophenyl-imidazole p38α MAPK, CK1δ and JAK2 kinase inhibitors

The synthesis and structure–activity relationships of novel 4-(4′-fluorophenyl)imidazoles as selective p38α MAPK, CK1δ and JAK2 inhibitors with improved water solubility are described. Microwave-assisted multicomponent reactions afforded 4-fluorophenyl-2,5-disubstituted imidazoles. Carboxylate and phosphonate groups were introduced via ‘click’ reactions. The kinase selectivity was influenced by the heteroaryl group at imidazole C-5 and the position of a carboxylic acid or tetrazole at imidazole C-2. For example, pyrimidines 15 and 34 inhibited p38α MAPK with IC50=250nM and 96nM, respectively. Pyridine 3 gave CK1δ inhibition with IC50=89nM and pyridin-2-one 31 gave JAK2 inhibition with IC50=62nM.

THU0127 Pharmacodynamics of A Novel JAK1 Selective Inhibitor in Rat Arthritis and Anemia Models and in Healthy Human Subjects

BackgroundAnti-cytokine therapies have become the mainstay of treatment for rheumatoid arthritis (RA) disease symptoms and can arrest disease progression. Despite numerous treatment options there are still many RA patients who...

ATP-mediated kinome selectivity: the missing link in understanding the contribution of individual JAK Kinase isoforms to cellular signaling.

Kinases constitute an important class of therapeutic targets being explored both by academia and the pharmaceutical industry. The major focus of this effort was directed toward the identification of ATP competitive inhibitors. Although it has long been recognized that the intracellular concentration of ATP is very different from the concentrations utilized in biochemical enzyme assays, little thought has been devoted to incorporating this discrepancy into our understanding of translation from enzyme inhibition to cellular function. Significant work has been dedicated to the discovery of JAK kinase inhibitors; however, a disconnect between enzyme and cellular function is prominently displayed in the literature for this class of inhibitors. Herein, we demonstrate utilizing the four JAK family members that the difference in the ATP Km of each individual kinase has a significant impact on the enzyme to cell inhibition translation. We evaluated a large number of JAK inhibitors in enzymatic assays utilizing either 1 mM ATP or Km ATP for the four isoforms as well as in primary cell assays. This data set provided the opportunity to examine individual kinase contributions to the heterodimeric kinase complexes mediating cellular signaling. In contrast to a recent study, we demonstrate that for IL-15 cytokine signaling it is sufficient to inhibit either JAK1 or JAK3 to fully inhibit downstream STAT5 phosphorylation. This additional data thus provides a critical piece of information explaining why JAK1 has incorrectly been thought to have a dominant role over JAK3. Beyond enabling a deeper understanding of JAK signaling, conducting similar analyses for other kinases by taking into account potency at high ATP rather than Km ATP may provide crucial insights into a compound's activity and selectivity in cellular contexts.

Sustained inhibition of STAT5, but not JAK2, is essential for TKI-induced cell death in chronic myeloid leukemia.

Kinase inhibitors block proliferative signals in BCR-ABL1+ leukemic cells, but their capacity to induce apoptosis is poorly understood. Initial studies suggested that very brief exposure to kinase inhibitors was sufficient to induce apoptosis in chronic myeloid leukemia (CML) cells. However, flaws in this experimental model have subsequently been identified, leading to the conclusion that apoptosis only occurs with sustained low-level kinase inhibition. Thus, the minimum duration of complete kinase inhibition required to commit CML cells to death is unknown. Here we confirm that <1 h is insufficient to induce significant commitment to death in BCR-ABL1+ cell lines and in primary CD34+ progenitor cells, and establish that commitment to cell death only occurs if kinase inhibition is maintained for 4 h or more. Remarkably, signal transducer and activator of transcription 5 (STAT5) inhibition in combination with transient (<1 h) tyrosine kinase inhibitor (TKI) exposure proved lethal for CML progenitors, despite the reactivation of Bcr-Abl after 1 h. JAK kinase inhibition did not induce cell death in combination with transient TKI exposure. Thus, STAT5 appears to be a critical determinant of the time-dependent sensitivity of CML progenitor cells to TKI treatment in a Bcr-Abl-dependent, but JAK-independent, manner. We conclude that combining kinase inhibition with STAT5 inhibition represents a promising therapeutic approach in BCR-ABL1+ leukemias.

Molecular Pathways: Molecular Basis for Sensitivity and Resistance to JAK Kinase Inhibitors

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.

Erratum: Specificity and mechanism-of-action of the JAK2 tyrosine kinase inhibitors ruxolitinib and SAR302503 (TG101348)

Correction to: Leukemia (2014) 28, 445–448; doi:10.1038/leu.2013.205; published online 19 July 2013 In the original publication of the article, the authors have inadvertently used a structural variant of the JAK2 kinase inhibitor SAR302503 (TG101348) for the molecular dynamics simulations shown in Figure 1 (right panels).

Rapid Molecular Diagnosis Of JAK2V617F Negative MPN By Targeted Deep Sequencing Using The Ion Torrent PGM

Myeloproliferative neoplasms (MPN) are a heterogeneous group of blood disorders characterized by excess production of mature blood cells, increased risk of thrombotic complications and slow progression to myelofibrosis or, less often, leukemia. Activation of the JAK-STAT signaling pathway is a common underlying feature of these diseases and JAK kinase inhibitors are efficacious in the more advanced forms of disease. Most cases of polycythemia vera (PV) and approximately 60% of essential thrombocythemia (ET) and primary myelofibrosis (MF) harbor a point mutation in JAK2 (V617F) which leads to constitutive JAK-STAT signaling and factor independent cell growth. The remaining 40% of cases of MF and ET harbor a broad range of mutations in many genes including those involved in cytokine receptor signaling, other components or the JAK-STAT pathway or epigenetic regulators. This poses a challenge for rapid molecular diagnosis. Also, since ET is essentially a diagnosis of exclusion of reactive causes of thrombocytosis, many cases of chronic ‘ET' may not be clonal hematological neoplasms but reactive conditions.We have developed a rapid deep sequencing pipeline to detect mutations in 65 genes which have been implicated in MPN through previous reports of human mutations, mouse models of MPN, or other known components of hematopoietic cytokine receptor signaling. We used 10ng of DNA from blood to amplify and sequence all the exons of these 65 genes using Ampliseq and Ion Torrent PGM. Using 318 PGM chips and 8-fold multiplexing we achieved on average 200 fold coverage of the target exome. The bioinformatics of SNP validation and rapid generation of reports will be presented. From a pilot study of 30 cases referred for molecular diagnosis, we have detected the likely causative mutation in approximately 80% of ET and MF where JAK2 is wild type. Many of these mutations are known to be causative in MPN, including those in MPL, ASXL1, SET2, SH2B3 (LNK), EZH2, CBL, DNMT3A and other genes. We have identified a novel inherited mutation in a family with MPN and validated it in BAF3 factor-independency assays. We have identified further novel mutations in JAK3, EED, DNMT3A, APC and two phosphatases involved in silencing activated JAK-STAT pathway components. The biological significance of these is under investigation and progress will be reported. In many cases we find evidence for clonal evolution involving secondary mutations in epigenetic modifying proteins on top of driver mutations in the JAK-STAT pathway.In short, targeted exome re-sequencing using Ampliseq and Ion Torrent PGM provides a rapid and relatively cheap method for molecular diagnosis and characterization of most cases of MPN. Disclosures:Perkins:Novartis Oncology: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Combined Therapy With BRD4 Antagonist and JAK Inhibitor Is Synergistically Lethal Against Human Myeloproliferative Neoplasm (MPN) Cells

Following therapy with type I JAK kinase inhibitors that exert activity only against the active JAK kinase, a lack of complete clinical or molecular response due to incomplete elimination of the MPN clone strongly supports the rationale to develop novel combinations against advanced MPN. Recent studies have demonstrated that despite continuing JAK inhibitor treatment, persistent JAK2 phosphorylation and the resulting heterodimeric JAK2 signaling may be responsible for JAK inhibitor-insensitive MPN persisters and resistance to therapy. In the present studies, we determined the activity of the BET (bromodomain and extraterminal) protein BRD4 antagonists JQI or I-BET151 with or without JAK inhibitors SAR302503 (SAR) and ruxolitinib (Rux) against human MPN cells that are sensitive or exhibit resistance to JAK inhibitors. BRD4 binds to acetylated lysines on the histone proteins, helps assemble transcriptional co-regulators at the target gene promoters and enhancers, and regulates the expression of important oncogenes, e.g., c-MYC, CDK6, BCL-2 and IL7R. Treatment with the BET protein inhibitor JQ1 disrupts the binding of the bromodomain of BRD4 to acetyl lysines in the chromatin associated with the oncogenes, thereby depleting their expression and inducing apoptosis of human leukemia cells. Our studies here demonstrated that JQ1 (250 to 1000 nM) but not its inactive enantiomer (R-JQ1) dose-dependently induced apoptosis of the MPN HEL92.1.7 (HEL) and UKE1 cells, as well as of CD34+ primary MPN cells harvested from the peripheral blood of patients with advanced MF. This was associated with decreased binding of BRD4 and RNA Pol II, by ChIP-qPCR analyses, to the promoters of c-MYC, BCL-2, CDK6 and IL7 receptor (IL7R), concomitantly with marked attenuation of the c-MYC, BCL-2, CDK6 and IL7R, but induction of p21 mRNA expression in HEL and UKE1 cells. I-BET151 (100 to 2000 nM) also exerted similar effects in MPN cells. JQ1 treatment also depleted the protein levels of p-JAK2, p-STAT5, p-STAT3, PIM1, BCL-2, BCL-xL, MCL and CDK4/6, while inducing p21 protein levels in the MPN cells. Following engraftment of NOD/SCID mice with HEL cell xenograft, co-treatment with JQ1 (50 mg/kg/day, formulated in 10% 2-hydroxypropyl-β-cyclodextrin, administered IP daily x 5 days, per week x 3 weeks) and SAR (100 mg/kg/day, PO daily x 5 days per week x 3 weeks) versus treatment with vehicle control, or JQ1 or SAR alone, resulted in significant in vivo attenuation of c-MYC and BCL-2 levels in the harvested MPN cells from the mice (p < 0.01). Importantly, JAK inhibitor (SAR and Rux)-resistant HEL cells (> 10-fold resistant to Rux) created under the selection pressure of a continuous exposure to JAK inhibitor (HEL/JIR) (Clin Cancer Res 2011;17:7347) remained sensitive to JQ1-induced apoptosis. JQ1 was also equally effective in inducing apoptosis of primary MPN cells harvested from patients pre- and post-JAK kinase inhibitor therapy. JQ1 treatment attenuated the levels of c-MYC, p-STAT5, PIM1, CDK6 and BCL-2 in HEL/JIR cells. Co-treatment with JQ1 or I-BET151 and the JAK kinase inhibitor SAR or Rux synergistically induced apoptosis of the cultured HEL and UKE1 cells, with concomitant greater attenuation of the p-JAK2, p-STAT5, p-STAT3, PIM1, BCL-2, BCL-xL, MCL and CDK4/6 levels. Collectively, these findings demonstrate that BRD4 antagonists exhibit potent activity against cultured and primary human MPN cells expressing JAK2V617F. These findings also highlight the novel and synergistic activity of the combination of BRD4 antagonist and JAK inhibitor against human MPN cells, and support the rationale for in vivo testing of the combination against JAK inhibitor-insensitive MPN persisters that exhibit resistance to the first generation of JAK inhibitors. Disclosures:No relevant conflicts of interest to declare.

The CSF3R T618I Mutation Found In Chronic Neutrophilic Leukemia Removes An O-Linked Glycosylation Site and Increases Receptor Dimerization

The CSF3R T618I Mutation Found In Chronic Neutrophilic Leukemia Removes An O-Linked Glycosylation Site and Increases Receptor Dimerization

Use of ω-Transaminase Enzyme Chemistry in the Synthesis of a JAK2 Kinase Inhibitor

ω-Transaminase enzyme chemistry provides an excellent methodology to build synthetically useful chiral amines from their corresponding ketones. An application of this methodology, providing a long-term commercial manufacturing route to a JAK2 kinase inhibitor, is reported herein.

78 : Therapeutic inhibition of Gp130/Jak/Stat3-dependent cytokine signaling suppresses WNT-dependent colon cancer formation

The initiating mutations in the majority of sporadic and familial colorectal cancers (CRC) occur in the tumor suppressor gene APC and result in nuclear accumulation of β -catenin associated with aberrant activation of the WNT pathway. Meanwhile, a tightly controlled burst of WNT signalling is required for regeneration of the intestinal epithelium after injury and ensures homeostasis. Since identification of therapeutic WNT pathway inhibitors remains challenging, we explored whether genetic interference with cytokine-dependent gp130/Jak/Stat3 signaling affects the intestine’s response to oncogenic WNT signaling. In situations of chronic inflammation and excessive secretion of interleukin- (IL) 6-family cytokines, we have previously shown that gp130 engagement simultaneously activates Stat3 and the mTOR complex 1, and that the mTorc1-specific inhibitor RAD001 potently suppressed initiation and progression of colitis-associated CRC (Thiem et al., J Clin Invest (2013) 123:767). Here we describe that partial genetic suppression of either Stat3 expression, or of its gp130 receptor-dependent activation, prevents tumorigenesis in mouse models of sporadic and of inherited CRC. Likewise, therapeutic administration of the Jak-kinase inhibitors effectively blocked de novo tumor initiation and progression in these models. Surprisingly, gp130/Jak/Stat3 pathway inhibition selectively impaired the growth of mutant APC mouse tumors and human CRC cell xenografts without affecting homeostasis of the non-neoplastic intestinal epithelium. We identify Stat3-dependent Bmi1 induction and the resulting repression of p16 and p21 as the mechanism by which the gp130/Jak/Stat3 pathway modulates the cellular response to aberrant WNT signaling. Meanwhile, a large proportion human CRC harbours coinciding WNT- and STAT3 gene activation signatures. Interference with the gp130/Jak/Stat3 pathway is therefore readily exploitable for the therapeutic treatment of CRC.

Hwanggeumchal sorghum extract enhances BMP7 and GH signaling through the activation of Jak2/STAT5B in MC3T3-E1 osteoblastic cells

Sorghum is a principal cereal food in a number of parts of the world and is critical in folk medicine in Asia and Africa. However, its effects on bone are unknown. Growth hormone (GH) is a regulator of bone growth and bone metabolism. GH activates several signaling pathways, including the Janus kinase (Jak)/signal transducer and activator of transcription (STAT) pathways, thereby regulating expression of genes, including insulin‑like growth factor (IGF)‑1. Bone morphogenetic proteins (BMPs) induce the differentiation of cells of the osteoblastic lineage, increasing the pool of IGF‑1 target cells, the mature osteoblasts. In the present study, the effects of Hwanggeumchal sorghum extracts (HSE) on GH signaling via the Jak/STAT pathway in osteoblasts were investigated. HSE was not observed to be toxic to osteoblastic cells and increased the expression of BMP7 and GH‑related proteins, including STAT5B, p‑STAT5B, IGF‑1 receptor (IGF-1R), growth receptor hormone (GHR) and Jak2 in MC3T3‑E1 cells. In addition, HSE increased BMP7 and GHR mRNA expression in MC3T3‑E1 cells. The expression of HSE‑induced BMP7 and GHR was inhibited by AG490, a Jak2 kinase inhibitor. The observations indicate that HSE‑induced signaling is similar to GH signaling via the GHR‑Jak2 signaling axis. Using small interference RNA (siRNA) analysis, STAT5B was found to play an essential role in HSE‑induced BMP7 and GH signaling in MC3T3‑E1 cells. Results of the current study indicate that HSE promotes bone growth through activation of STAT5B.

Sensitivity and resistance of JAK2 inhibitors to myeloproliferative neoplasms

The discovery of activating mutations in JAK2 and MPL in a majority of patients with myeloproliferative neoplasms (MPN) has led to the rapid clinical development of several JAK kinase inhibitors. Of these, the JAK1/2 inhibitor, ruxolitinib (INCB018424, Incyte Corporation) was recently approved for the treatment of patients with myelofibrosis (MF). JAK inhibitors have effectively reduced splenomegaly and high cytokine levels in patients leading to improvements in quality of life. However, they have not been successful in eliminating the mutant clone in a majority of patients. In vitro studies using saturation mutagenesis screens have revealed several mutations in JAK2 that confer resistance to JAK inhibitors. Nevertheless, these mutations have not been identified so far in JAK inhibitor-treated patients. A recent study from our laboratory demonstrated that chronic JAK kinase inhibition leads to JAK inhibitor persistence via transphosphorylation of JAK2 through other JAK kinase family members. This phenomenon is seen in cell lines, mouse models and patient samples. The JAK inhibitor persistent cells, however, still remain JAK2 dependent and therefore combination therapies that target JAK2 and other components of the JAK-STAT pathway along with JAK inhibitors may provide additional benefits and improve clinical outcomes in these patients.

Jak2V617F myeloproliferative neoplasm stem cells and interferon-alpha

The myeloproliferative neoplasms (MPNs) are clonal disorders of hematopoiesis that arise as a result of aberrant activation of tyrosine kinases and result in the proliferation and accumulation of mature myeloid cells in the blood, bone marrow and spleen. The prototypical MPN, chronic myeloid leukemia (CML) is caused by constitutive activation of ABL kinase occurring as a result of the BCR-ABL fusion. Importantly, targeted therapy to inhibit ABL kinase signaling, using imatinib, induces durable clinical responses in the majority of CML patients. The molecular pathogenesis of the BCR-ABL negative MPNs, polycythemia vera (too many red cells), essential thrombocythemia (too many platelets) and myelofibrosis, was elucidated in 2005 with the discovery of JAK2V617F, an activating mutation in the non-receptor tyrosine kinase, JAK2 [1]. The JAK2V617F mutation is present in more than 95% patients with polycythemia vera, and approximately 50% patients with essential thrombocythemia and myelofibrosis. In advanced disease, MPN may transform to acute leukaemia and this is associated with a dismal prognosis. In MPN, JAK2V617F is found in long-term hematopoietic stem cells (LT-HSCs), a population of rare quiescent cells present in the bone marrow and capable of long-term self-renewal. These somatically mutated LT-HSCs, referred to as MPN-propagating cells or MPN stem cells, maintain the MPN throughout the life of the patient and can serially transplant disease in animal models of Jak2V617F-mediated MPN [2, 3]. To definitively cure MPN will require complete eradication of the MPN disease-maintaining stem cell population. In contrast to the efficacy of imatinib in achieving molecular remissions in CML, JAK2 kinase inhibitors have shown only modest benefit in the treatment of myelofibrosis, namely in reducing splenomegaly and improving constitutional symptoms. Furthermore, the lack of an effect of the JAK2 inhibitors on reducing JAK2V617F allele burden in myelofibrosis patients [4] and the demonstration that MPN-propagating cells are insensitive to JAK2 kinase inhibition in vivo [2] suggests that MPN stem cells do not require JAK2 signaling for survival and that JAK2 inhibitor monotherapy will not cure MPN patients. The clinical efficacy of long-acting pegylated IFNα in MPN has renewed interest in understanding the mechanism of action of IFNα treatment in JAK2V617F-mediated MPN. Prolonged IFNα treatment results in normalization of blood counts in most patients with polycythemia vera, reduces JAK2V617F allele burden and in 15-40% of patients, the JAK2V617F mutant clone is rendered undetectable to sensitive molecular assays[5, 6]. These clinical results suggest that IFNα eradicates JAK2V617F-mutant LT-HSC, however the mechanism by which molecular remissions are achieved in MPN patients in response to treatment with IFNα have not been well defined. Figure 1 MPN stem cells are responsible for disease initiation and propagation in vivo To determine the efficacy of IFNα therapy on Jak2V617F-positive MPN stem cells in vivo, we used a murine model of Jak2V617F-mediated MPN [7]. We found that prolonged treatment of Jak2V617F mice with IFNα reversed the myeloproliferation phenotype (normalizing hemoglobin and white cell count and reducing extramedullary hematopoiesis). Importantly, we observed selective depletion of Jak2V617F over WT LT-HSC and this correlated with an inability of IFNα treated mice to propagate MPN into transplanted recipients. IFNα can directly activate the cell cycle in quiescent wild-type (WT) LT-HSCs and induce HSC proliferation in vivo [8]. We found that IFNα treatment induced enhanced cell-cycle activation of Jak2V617F-mutant LT-HSCs, as compared to WT LT-HSCs, explaining this preferential depletion over time. Using Ifnar1 knock-out mice (lacking the type 1 interferon receptor), we showed that the effects of IFNα on cell cycle are specifically mediated through type 1 interferon signaling and are intrinsic to Jak2V617F-mutant LT-HSCs. Using gene expression analysis we found an enrichment of cell cycle genes in Jak2V617F-mutant LT-HSC, both at baseline and after IFNα treatment, providing a potential explanation for their preferential sensitivity to IFNα treatment. This work suggests that IFNα achieves molecular remissions in JAK2V617F-positive MPN patients through the activation and consequent depletion of quiescent MPN disease-propagating cells. What are the clinical implications of this work? Firstly, these data support the rational design of clinical trials to test combinatorial therapeutic approaches using IFNα (to target quiescent MPN-stem cells) together with JAK2 kinase inhibitors or cytotoxic chemotherapy (to target actively cycling progenitor populations). Plausibly, the depletion of MPN stem cells in polycythemia vera could alter the natural history of the disease, through eliminating the malignant clone and thus preventing fibrotic and/or leukemic transformation, both devastating complications of MPN. Currently, IFNα is far from an ideal drug, and few patients are able to tolerate prolonged treatment due to its side effects. We have identified a number of novel targets that are differentially regulated in Jak2V617F and WT LT-HSCs following IFNα treatment. In ongoing studies, we are working to validate these targets with the goal of developing more specific, less toxic IFNα derivatives capable of selectively eliminating MPN stem cells, while sparing normal LT-HSCs and other organ systems.

Molecular pathways: Jak/STAT pathway: mutations, inhibitors, and resistance.

Aberrant activation of the JAK/STAT pathway has been reported in a variety of disease states, including inflammatory conditions, hematologic malignancies, and solid tumors. For instance, a large proportion of patients with myeloproliferative neoplasms (MPN) carry the acquired gain-of-function JAK2 V617F somatic mutation. This knowledge has dramatically improved our understanding of the pathogenesis of MPNs and has facilitated the development of therapeutics capable of suppressing the constitutive activation of the JAK/STAT pathway, now recognized as a common underlying biologic abnormality in MPNs. Ruxolitinib is an oral JAK1 and JAK2 inhibitor that has recently been approved for the treatment of myelofibrosis and has been tested against other hematologic malignancies. A series of agents with different specificities against different members of the JAK family of proteins is currently undergoing evaluation in clinical trials for patients with MPNs, lymphoma, and solid tumors such as breast or pancreatic cancer. Despite the significant clinical activity exhibited by these agents in myelofibrosis, some patients fail to respond or progress during JAK kinase inhibitor therapy. Recent reports have shed light into the mechanisms of resistance to JAK inhibitor therapy. Several approaches hold promise to overcome such resistance.