scholarly journals Heterodimeric JAK-STAT Activation As a Mechanism of Persistence to JAK2 Inhibitor Therapy

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 122-122
Author(s):  
Neha Bhagwat ◽  
Priya Koppikar ◽  
Outi Kilpivaara ◽  
Taghi Manshouri ◽  
Mazhar Adli ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Clinical Response ◽  
Cell Lines ◽  
Jak Inhibitor ◽  
Epigenetic Alterations ◽  
Jak2 Inhibitor ◽  
In Trans ◽  
Mutant Cells ◽  
Jak2 Inhibition ◽  
Jak2 Inhibitors

Abstract Abstract 122 Although JAK2 inhibitor therapy improves MPN-associated splenomegaly and systemic symptoms, JAK2 inhibitor treatment does not significantly reduce or eliminate the MPN clone in most MPN patients. We therefore sought to characterize mechanisms by which MPN cells can persist despite chronic JAK2 inhibition. We performed saturation mutagenesis followed by next-generation sequencing in JAK2 mutant cells exposed to two different JAK2 inhibitors, INCB18424, a dual JAK1/JAK2 inhibitor, and JAK Inhibitor I, a pan-JAK inhibitor. Although we were able to identify candidate resistance alleles, these alleles were present in less than 50% of the total population. These data and the clinical experience with JAK2 inhibitors suggest that the failure of JAK2 inhibitors to reduce disease burden is not due to acquired drug resistance but rather due to persistent growth and signaling in the setting of chronic JAK2 kinase inhibition. We therefore generated JAK2/MPL mutant JAK2-inhibitor persistent (JAKper) cell lines (SET-2, UKE-1, Ba/F3-MPLW515L). JAKper cell lines are able to survive and proliferate in the presence of JAK2 inhibitors including JAK Inhibitor I, INCB18424 and TG101348 without acquiring second-site resistance alleles and are also insensitive to other JAK inhibitors. Signaling studies revealed JAK-STAT signaling was reactivated in persistent cells at concentrations of inhibitor that completely abrogated signaling in naïve cells, and JAK2 phosphorylation was reactivated in JAK inhibitor persistent cells consistent with reactivation of the JAK-STAT pathway in JAKper cells despite inhibitor exposure. We hypothesized that JAK2 may be activated in trans by other JAK kinases, and found an increased association between activated JAK2 and JAK1/TYK2 consistent with activation of JAK2 in trans by other JAK kinases in JAKper cells. We next assessed whether JAK inhibitor persistence was reversible. Withdrawal of JAK2 inhibitors from JAKper cells for 2 weeks led to resensitization such that JAKper resensitized cells were now sensitive to different JAK2 inhibitors regardless of previous exposure. Resensitization was associated with reversal of heterodimerization and loss of transactivation of JAK2 by JAK1 and TYK2. The reversible nature of JAK inhibitor persistence led us to hypothesize epigenetic alterations are responsible for JAK inhibitor insensitivity in JAKper cells; we observed increased expression of JAK2 at the mRNA and protein level in JAK2 inhibitor persistent cells compared to parental as well as resensitized cells. ChIP-PCR analysis of the JAK2 locus revealed a significant increase in H3K4-trimethylation and a reduction in H3K9 trimethylation in persistent cells compared to parental cells consistent with a change to a more active chromatin state at the JAK2 locus and increased JAK2 mRNA expression in persistent cells. We next assessed whether the same phenomenon of JAK2 inhibitor persistence was observed in vivo. In a MPLW515L-mutant murine bone marrow transplant model of primary myelofibrosis, we observed increased JAK2 expression, increased JAK2 phosphorylation and JAK-inhibitor induced association between JAK1 and JAK2 in hematopoietic cells from INCB18424 treated mice. We next extended our findings to samples from patients treated with INCB18424. We identified 5 patients who had a significant clinical response and 5 patients without a significant clinical response as assessed by spleen size and JAK2V617F allele burden responses and measured JAK2 granulocyte mRNA expression before and during INCB18424 treatment. We found that JAK2 mRNA levels significantly increased in INCB18424 nonresponders compared to responders (p=0.05) suggesting this phenomenon is observed in cell lines, mouse models and primary samples. Finally, we investigated whether JAKper cells remain JAK2 dependent. Studies with shRNA targeting JAK2 and pharmacologic studies using Hsp90 inhibitors that degrade JAK2 protein demonstrate that JAK2 inhibitor persistent cells remain dependent on JAK2 protein expression. Our data indicate that JAK2/MPL mutant cells persist in the presence of JAK2 kinase inhibitors through epigenetic alterations which reactivate signaling in persistent cells, and that therapies which lead to JAK2 degradation can be used to inhibit signaling and improve outcomes in patients with persistent disease despite chronic JAK2 inhibition. Disclosures: Verstovsek: Incyte Corporation: Research Funding.

scholarly journals Acquired Resistance to JAK Inhibitors in Calr-Mutated Myeloproliferative Neoplasms

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2970-2970
Author(s):  
Ken-Hong Lim ◽  
Yu-Cheng Chang ◽  
Yi-Hao Chiang ◽  
Huan-Chau Lin ◽  
Ling Huang ◽  
...  
Keyword(s):  
Cell Lines ◽  
Acquired Resistance ◽  
Jak Inhibitor ◽  
Secondary Mutation ◽  
Wild Type ◽  
Jak Inhibitors ◽  
Jak2 Inhibitor ◽  
Stat Signaling ◽  
Calr Mutations ◽  
Mutant Cells

Background: Calreticulin (CALR) mutations are one of the major driver mutations in BCL-ABL1-negative myeloproliferative neoplasm (MPN) and are frequently detected in JAK2/MPL-unmutated essential thrombocythemia and primary myelofibrosis. Mutant CALR activates JAK-STAT signaling through an MPL-dependent mechanism to mediate pathogenic thrombopoiesis in MPNs. Although JAK inhibitors such as ruxolitinib can provide important clinical benefits to MPN patients including those harboring CALR mutations, JAK inhibition does not preferentially target the MPN clone and acquired resistance develops over time. We aimed to characterize the mechanisms of acquired resistance to JAK inhibitors in CALR-mutated hematopoietic cells and to screen for novel therapeutic approaches specifically target CALR-mutant cells in this study. Methods: UT-7/TPO-derived cell lines expressing wild-type and type 1 and type 2 mutant CALR (CALRdel52 and CALRins5) were kindly provided by Drs. Komatsu and Araki. JAK2-inhibitor-resistant cells were generated by co-cultured with ruxolitinib and fedratinib (TG101348, a JAK2-selective inhibitor). JAK-STAT signaling was evaluated by Western blot on CALR-wild-type and mutated cells exposed to JAK2 inhibitor compared to untreated cells. For the detection of acquired secondary mutations in CALR-mutated cells exposed to JAK2 inhibitor, whole exome sequencing (WES) was performed using the BGISEQ-500 Sequencing platform (BGI, Shenzhen, China) with the 2 x 100 bp paired-end protocol. Genome Analysis Toolkit was used for variation detection. Reads were aligned to human reference genome hg19 using BWA version 0.7.15. Targeted resequencing was performed on leukocytes from patients with MPN who had been treated with ruxolitinib. Screening with chemical libraries/novel compounds will be conducted on UT7/TPO-CALR cell lines. Results: Compared to the parental cells, ruxolitinib-resistant UT7/TPO-CALR mutant cell lines have developed significant cross resistance to other JAK inhibitor as shown in the cell viability study. Signalling downstream of JAK2 in all 3 inhibitor-naïve UT-7/TPO/CALR parental cell lines was inhibited by acute treatment of ruxolitinib as shown on Western blot. Whereas, constitutive JAK2 activation was observed in all 3 inhibitor-resistant UT-7/TPO/CALR cell lines. No change in the expression of Epo and MPL receptors in these cell lines was found. Interestingly, constitutive JAK3 activation was also seen in inhibitor-resistant UT-7/TPO/CALR cells in comparison with parental cells. These findings indicated the presence of transphosphorylation by JAK3 in inhibitor-resistant UT-7/TPO/CALR cell lines. In addition, the results of WES identified several acquired secondary mutations in 3 inhibitor-resistant UT-7/TPO/CALR cell lines including SH2B1, ABCC1, HOXB3 and KRTAP4-5. No acquired secondary mutation was identified in CALR and other genes involved in JAK-STAT signaling. Acquired secondary mutation will be screened in primary MPN patients' samples treated with JAK inhibitor. Type II JAK inhibitor such as BBT-594 has been shown to inhibit JAK activation and signaling in JAK-persistent/resistant cells. Conclusions: Our results confirmed that the in vitro efficacy of JAK2 inhibition on CALR-mutant cells. Our data also suggested that JAK2 transphosphorylation and acquired secondary mutations could be underlying mechanisms for acquired resistance to JAK inhibitors in CALR-mutated cells. Novel therapeutics approaches should be developed to overcome acquired resistance in CALR-mutated cells. Disclosures No relevant conflicts of interest to declare.

JAK2 Inhibitor Therapy in Chronic Myeloproliferative Neoplasms Ph(-): Hematologic, Clinical and Molecular Responses

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5059-5059
Author(s):  
Ana Esther Kerguelen Fuentes ◽  
Dolores Hernández-Maraver ◽  
Miguel Angel ◽  
Canales Albendea ◽  
Ana Rodriguez de la Rua
Keyword(s):  
Clinical Response ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Molecular Response ◽  
Spleen Size ◽  
Molecular Responses ◽  
Allele Burden ◽  
Jak2 Inhibitor ◽  
Chronic Myeloproliferative Neoplasms ◽  
Jak2 Inhibitors

Abstract Abstract 5059 JAK2 inhibitors are known to improve symptoms, to control myeloproliferation and to reduce splenomegaly in patients diagnosed with chronic myeloproliferative neoplasms (MPNs)Ph(-). However their ability to decrease the allele burden and achieve molecular responses is controversial. Objective: To evaluate hematologic, clinical and molecular responses according to the criteria of the European LeukemiaNet and European Myelofibrosis Network in 13 patients treated with JAK2 inhibitors. Material and Methods: We performed a prospective study in the Haematology Service of the Hospital La Pazbetween 1987 and 2012 in 13 patients diagnosed with NMP Ph (-) and treated with of JAK2 inhibitors: 5 secondary mylofibrosis (SFM)to homozygous polycythemia vera JAK (+), 4 SFM to essential thrombocythemias JAK (-), 2 primary myelofibrosis (one JAK (-) and one heterozygous JAK (+)) and 2 homozygous PV JAK (+) resistant to hydrea. The RT-PCR was performed at 6 or 12 months after the first determination of the allelic burden. Median follow-up was 3 months (1 – 15). A) Hematologic Response (HR): 3/5 SFM to PV(1)/TE JAK(-)(2) reached HR at 3 months of initiation of JAK2 inhibitor to 20mg/day. Molecular and clinical response were not evaluated. B) Clinical Response: Three patients had a reduction in the spleen size. Only one patient in the SFM group had a reduction in the spleen size (18 cm before the drug was commenced to 13. 7 cm) and the allele burden decrease from 55% to 23% after 5 months of therapy with JAK2 inhibitor at 25mg/12h (increase of 5mg/12h after 15 days of initiation of medication). 2/3 MFS to TE JAK(-) had a reduction from 15, 3 cm before the drug was commenced to 9 cm after 3 months of therapy with JAK2 inhibitor at 20 mg/12h. 3/3 MFP JAK(-) had a 6cm reduction in spleen size. Twenty cm splenomegaly was documented before starting JAK2 inhibitor to 15 mg/day. C) Molecular Response: 2/5 SFM to PV decreased the previous allele burden value. One patient decreased by 25% the previous allele burden value (99. 28%) at 6 months of JAK2 inhibitor. Second patient decreased by 13% the previous allele burden value (55%) at 6 months of starting JAK2 inhibitor to 25 mg/day. In 1/2 PV, the previous allele burden value (93. 17%) decreased by 11. 4% at 6 months of starting JAK2 inhibitor at 100mg/24h. D) Lack of response and disease progression: One patient with SMF secondary to JAK 2 (-) ET had dose reductions from 20 mg twice a day secondary to grade IV thrombocytopenia and renal toxicity. Patient finally developed acute leukemia. Conclusions: Our study confirms that JAK2 inhibitors reduce splenomegaly in MPNs JAK(-)and JAK(+). Prospective studies with an adequate sample size are necessary to demonstrate whether splenomegaly and symptom reductions achieved with inhibition of JAK2 could be associated to decrease the allele burden and achieve molecular responses in MPNs JAK(+). Disclosures: No relevant conflicts of interest to declare.

A New Prognostic Model for Response in Myelofibrosis Patients Treated with JAK2 Inhibitors: A Study from Three US Academic Centers

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1842-1842 ◽  
Author(s):  
Menghrajani Kamal ◽  
Philip S. Boonstra ◽  
Alissa A. Weber ◽  
Cecelia Perkins ◽  
Krisstina L. Gowin ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Clinical Response ◽  
Bone Marrow Biopsy ◽  
Risk Score ◽  
Initial Dose ◽  
Research Funding ◽  
Spleen Size ◽  
Jak2 Inhibitor ◽  
European Consensus ◽  
Jak2 Inhibitors

Abstract Background: JAK2 inhibitors have been shown to improve symptoms and produce durable reductions in splenomegaly in patients with myelofibrosis (MF), and ruxolitinib has been shown to improve survival in MF patients (Cervantes et al., 2013; Verstovsek et al., 2013). Current prognostic models such as DIPSS plus (Gangat et al., 2011) predict survival in MF based on clinical, laboratory, and cytogenetic information, but their value in predicting clinical response or survival during treatment with JAK2 inhibitors remains unknown. We hypothesized that clinical features such as bone marrow fibrosis and splenomegaly may have independent effects on therapy response. Therefore, we conducted a retrospective analysis to create a new model to risk stratify patients with respect to their likelihood of responding to oral JAK2-inhibitor therapy. Methods: We studied a cohort of 203 patients with bone marrow biopsy-proven MF seen at University of Michigan, Stanford University, and Mayo Clinic in Scottsdale, AZ. These patients were all treated with ruxolitinib or an experimental JAK2 inhibitor. Our primary endpoint was defined as IWG-MRT criteria for splenic response by palpation (Tefferi et al., 2013). Response in patients with spleen size of less than 5 cm was defined as complete resolution of splenomegaly. Of the 203 patients studied, splenic response was evaluated after 3 months of therapy in 167 patients and after 6 months of therapy in 138 patients; 127 patients were in both groups. A logistic regression was performed to identify factors that would predict clinical response. Results: The following characteristics were significantly associated with spleen response at 3 and 6 months: initial spleen size, European consensus criteria grading of MF on bone marrow biopsy, initial DIPSS plus score, and initial WBC count. Cellularity on marrow biopsy was not significant. We enriched a baseline logistic model of initial dose of oral JAK2 therapy and DIPSS plus score with additional prognostic factors. We found the following clinical characteristics to be jointly associated with splenic response: normalized initial dose of oral JAK2 inhibitor, initial spleen size, DIPSS plus score, degree of fibrosis by European consensus criteria. Duration of disease from time of diagnosis to time of treatment initiation was not prognostic for splenic response. We used this model to calculate the probability of splenic response based on a risk score: Risk score = –1.18(Dose) + 0.09(Initial Spleen Size in cm) + 0.20(DIPSS-plus Points) +0.92 (if fibrosis is MF-3). The probability at 6 months can then be calculated from the risk score as follows: [1/(1+e(risk score-2.6))]. The figure demonstrates the prognostic gain of our model over a model based on DIPSS plus alone. Conclusion: With this observational study, we propose a predictive model which may serve as a clinical tool to identify which patients are most likely to benefit from JAK2 inhibitor-based therapies. Further validation in independent data sets will be required before this model can be more widely applied. Disclosures Mesa: Incyte Corporation, CTI, NPS Pharma, Inc., Gilead Science Inc., Celgene: Research Funding. Gotlib:Incyte: Consultancy, Honoraria, Research Funding, Travel Reimbursement Other; Gilead: Research Funding; Sanofi: Research Funding; Novartis: Research Funding, Travel Reimbursement, Travel Reimbursement Other. Talpaz:ARIAD Pharmaceuticals, Inc., BMS, Sanofi, Incyte, Pfizer: Research Funding.

scholarly journals CYT387, a novel JAK2 inhibitor, induces hematologic responses and normalizes inflammatory cytokines in murine myeloproliferative neoplasms

Blood ◽  
2010 ◽  
Vol 115 (25) ◽  
pp. 5232-5240 ◽  
Author(s):  
Jeffrey W. Tyner ◽  
Thomas G. Bumm ◽  
Jutta Deininger ◽  
Lisa Wood ◽  
Karl J. Aichberger ◽  
...  
Keyword(s):  
Cell Lines ◽  
Inflammatory Cytokines ◽  
Kinase Inhibitor ◽  
Myeloproliferative Neoplasms ◽  
Symptomatic Improvement ◽  
Janus Kinase ◽  
Jak2 Inhibitor ◽  
Cell Counts ◽  
Tyrosine Kinase 2 ◽  
Jak2 Inhibitors

Abstract Activating alleles of Janus kinase 2 (JAK2) such as JAK2V617F are central to the pathogenesis of myeloproliferative neoplasms (MPN), suggesting that small molecule inhibitors targeting JAK2 may be therapeutically useful. We have identified an aminopyrimidine derivative (CYT387), which inhibits JAK1, JAK2, and tyrosine kinase 2 (TYK2) at low nanomolar concentrations, with few additional targets. Between 0.5 and 1.5μM CYT387 caused growth suppression and apoptosis in JAK2-dependent hematopoietic cell lines, while nonhematopoietic cell lines were unaffected. In a murine MPN model, CYT387 normalized white cell counts, hematocrit, spleen size, and restored physiologic levels of inflammatory cytokines. Despite the hematologic responses and reduction of the JAK2V617F allele burden, JAK2V617F cells persisted and MPN recurred upon cessation of treatment, suggesting that JAK2 inhibitors may be unable to eliminate JAK2V617F cells, consistent with preliminary results from clinical trials of JAK2 inhibitors in myelofibrosis. While the clinical benefit of JAK2 inhibitors may be substantial, not the least due to reduction of inflammatory cytokines and symptomatic improvement, our data add to increasing evidence that kinase inhibitor monotherapy of malignant disease is not curative, suggesting a need for drug combinations to optimally target the malignant cells.

Preclinical Analyses Of The Chemical JAK2 Inhibitor, SAR302503, In Classical Hodgkin Lymphoma and Primary Mediastinal Large B-Cell Lymphoma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4230-4230 ◽  
Author(s):  
Yansheng Hao ◽  
Bjoern Chapuy ◽  
Stefano Monti ◽  
Heather Sun ◽  
Scott J. Rodig ◽  
...  
Keyword(s):  
Cell Lines ◽  
Copy Number ◽  
B Cell Lymphoma ◽  
Dependent Manner ◽  
Jak2 Inhibitor ◽  
Large B Cell Lymphoma ◽  
Xenograft Models ◽  
Jak2 Inhibition

Abstract Classical Hodgkin lymphoma (cHL) and primary mediastinal large B-cell lymphoma (MLBCL) are diseases which share certain clinical, pathologic and genetic features. We previously characterized chromosome 9p24 amplification as a disease-specific structural alteration in cHL and MLBCL and identified the immunoregulatory genes, PD-L1 and PD-L2, and JAK2 as key targets of the 9p24 amplification. In a panel of informative cHL and MLBCL cell lines with defined 9p24 copy number, JAK2 amplification increased JAK2 protein expression and activity and enhanced sensitivity to chemical JAK2 inhibition with commercially available tool compounds. Given the importance of JAK/STAT signaling as a survival pathway in primary cHL and MLBCL and the pathway’s additional role in augmenting PD-1 ligand transcription, we postulated that JAK2 was a promising rational therapeutic target in cHLs and MLBCLs with 9p24 amplification. SAR302503 (fedratinib), formerly TG101348, is a selective, potent and oral JAK2 inhibitor that has demonstrated clinical activity in myeloproliferative disorders with activating JAK2 mutations. For these reasons, we have evaluated the preclinical activity of SAR302503 in in vitro and in vivo model systems of cHL and MLBCL with known 9p24/JAK2 copy number. In in vitro analyses, SAR302503 inhibited the cellular proliferation of cHL and MLBCL cell lines and induced their apoptosis. Of note, there was an inverse correlation between 9p24/JAK2 copy number and the EC50 of SAR302503, indicating that cHLs and MLBCLs with JAK2 amplification exhibited increased sensitivity to chemical JAK2 inhibition. In this series of cHLs and MLBCLs of defined JAK2 copy number, JAK2 copy gain was associated with higher baseline phosphorylated JAK2 (pJAK2) and increased abundance of phosphorylated STAT family members including pSTAT1, pSTAT3 and pSTAT6. Consistent with the copy number-dependent anti-proliferative effects of SAR302503, the compound decreased pJAK2, pSTAT1, 3 and 6 in a copy-number dependent manner in cHLs and MLBCLs. These effects were apparent within 2 hours of SAR302503 treatment. We next developed a comprehensive phosphoJAK/STAT immunohistochemical signature to assess baseline pathway activity and sensitivity to targeted chemical JAK2 inhibition. In cHL cell lines with 9p24 amplification, SAR302503 treatment abrogated pJAK2, pSTAT1 and pSTAT3 immunohistochemical staining at early and late timepoints. Furthermore, chemical JAK2 inhibition decreased PD-L1 transcript abundance in 9p24-amplified cHL and MLBCL cell lines and reduced the abundance of downstream JAK2 targets including MYC and PIM1 in cHLs with high JAK2 copy number. After demonstrating the activity and specificity of SAR302503 in in vitro assays, we evaluated the JAK2 inhibitor in murine xenograft models of cHL and MLBCL with 9p24/JAK2 amplification (Karpas 1106 [MLBCL] and HDLM2 [cHL]). In systemic MLBCL (Karpas 1106) and subcutaneous cHL (HDLM2) xenograft models, tumor growth was monitored via bioluminescent imaging (Karpas 1106) and mass measurements (HDLM2) and SAR302503 treatment was begun following the establishment of ≅ 100 mm3 tumors. In both models, pSTAT3 was analyzed as a pharmacodynamic marker following 5 days of treatment and SAR302503-treated animals had markedly decreased tumor cell pSTAT3 expression. In the Karpas 1106 MLBCL murine model, SAR302503 treatment significantly prolonged overall survival (p = .0002). In the HDLM2 cHL xenograft model, the JAK2 inhibitor significantly decreased subcutaneous tumor growth (p< .0001) and pSTAT3 expression in tumor cells (p = .0004). Transcriptional profiling confirmed that the HDLM2 tumors from SAR302503-treated animals exhibited coordinately decreased expression of STAT3 targets and downregulation of a functionally validated JAK2 gene set. Taken together, these data indicate that SAR302503 specifically decreases cHL and MLBCL growth in a 9p24/JAK2 copy number-dependent manner in vitro and in vivo and highlight the utility of pSTAT3 immunohistochemistry as a biomarker of pathway responsiveness. Clinical evaluation of JAK2 pathway inhibition in patients with cHL and MLBCL with known 9p24/JAK2 copy number status is warranted. Disclosures: Hao: Sanofi: Research Funding. Off Label Use: Drug is not yet approved but is being evaluated in myelofibrosis. Shipp:Sanofi: Research Funding.

Jak2 Phosphorylates Tyr 177 of Bcr-Abl Activating the Ras and PI-3 Kinase Pathways and Maintains Functional Levels of Bcr-Abl in Chronic Myelogenous Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 39-39 ◽  
Author(s):  
Ajoy K. Samanta ◽  
Sandip N. Chakraborty ◽  
Xiaoping Sun ◽  
Ellen Schlette ◽  
Waldemar Priebe ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Cell Lines ◽  
Nude Mice ◽  
Kinase Inhibitor ◽  
Blast Crisis ◽  
Myelogenous Leukemia ◽  
Protein Levels ◽  
Jak2 Inhibition ◽  
Jak2 Inhibitors

Abstract Abstract 39 We have shown that Jak2 is activated in Bcr-Abl+ hematopoietic cells. Our findings indicate that Jak2 kinase inhibition overcomes drug-resistance in CML cells (Samanta et al., 2006). Recently, we showed that Jak2 inhibition deactivates the Lyn kinase in Bcr-Abl+ cells by inhibiting SET expression; SET inhibits the PP2A-SHP1 (Samanta et al., 2009). Because of the critical effects of Jak2 in Bcr-Abl+ cells, we explored the mechanism of Jak2 action in CML cells. Bcr-Abl is known to be phosphorylated on a number of tyrosine residues including tyrosine 177. Phosphorylation of Tyr 177 leads to binding to Grb2, activation of the SOS-Ras pathway, and activation of the PI-3 kinase through the Grb2-Gab2 pathway. Here, we show that Jak2 and not Bcr-Abl phosphorylates Tyr 177 of Bcr-Abl. Tyr 177 of Bcr-Abl (YVNV) has the Jak2 consensus target sequence (YxxV/L/I) (Argetsinger et al., 2004) as do a number of other Tyr residues in the Bcr portion of Bcr-Abl (b3a2). Inhibition of Jak2 by a specific Jak2 kinase inhibitor TG101209 (TargeGen Inc., San Diego, CA) and a new Jak2 kinase inhibitor (WP1193, synthesized by Dr. Priebe) in Bcr-Abl+ cells, but not inhibition of Bcr-Abl by imatinib mesylate (IM), rapidly reduced phosphorylation of Tyr 177 of Bcr-Abl in IM-sensitive and –resistant cell lines including K562, K562-R, Bcr-Abl+ 32D, Bcr-Abl+ BaF3, Bcr-Abl+T315I BaF3 cells and cells from blast crisis CML patients. Jak2 inhibitors but not IM also strongly inhibited the phosphorylation of a synthetic Bcr peptide containing Tyr 177 (and the surrounding Bcr sequences) catalyzed by either recombinant purified Jak2 or Jak2 immune complexes. Jak2 inhibition of Bcr-Abl+ 32D cells rapidly reduced Grb2 binding to Bcr-Abl, and diminished activation of Ras and PI-3 kinase pathways within two h. Importantly, knockdown of Jak2 with Jak2-specific siRNA also reduced levels of pTyr 177 Bcr-Abl and total Bcr-Abl protein. Rescue experiments reversed Jak2 knockdown and stimulated pTyr 177 levels. In whole cells the total levels of the Bcr-Abl protein also decreased upon Jak2 inhibition with the above chemical inhibitors but at a rate slower than the rate observed in the inhibition of the Tyr 177 phosphorylation of Bcr-Abl. In kinase assays with Bcr-Abl network complexes from Bcr-Abl+ cells, phosphorylation of Tyr 177 of Bcr-Abl was drastically reduced by Jak2 inhibition but Bcr-Abl protein levels remained stable. Reduction of Bcr-Abl protein levels in Bcr-Abl+ cells by Jak2 inhibition also caused a severe reduction of tyrosine phosphorylation of STAT5 and STAT3. We have shown by gel filtration column chromatography of lysates isolated from Bcr-Abl+ cells that Bcr-Abl is physically associated with the IL-3 receptor beta chain, Jak2, Akt and HSP90 in high molecular weight complexes. GSK3β, STAT5 and STAT3 also emerge from the column in the high molecular weight fractions and suggested that several high molecular weight complexes may exist. Nevertheless, incubation of Bcr-Abl+ cells with WP1193 caused disruption of these large molecular network complexes within three h. Nude mice, injected subcutaneously with IM-resistant K562-R cells developed solid tumors, which were reduced in weight by i.p. injection of WP1193 every 48 h at 30 mg/Kg over 10 days. WP1193 at a similar dosage strongly reduced leukemic and tumor effects of Bcr-Abl+ 32D cells expressing T315I Bcr-Abl injected i.v. into nude mice. WP1193 injections into nude mice did not cause significant toxicity over a 10 day period. In summary, Jak2 inhibitors TG101209/WP1193 rapidly reduced levels of pTyr 177 Bcr-Abl, reduced levels of Bcr-Abl protein and induced high levels of apoptosis in IM-sensitive and –resistant Bcr-Abl+ cell lines including T315I cells, CML cell lines, cells from CML patients in blast crisis. In addition Jak2 inhibition reduced tumor formation in mice injected with IM-resistant K562-R and T315I Bcr-Abl+ mouse cells. These findings establish: 1) Jak2 as a major driver of Bcr-Abl signaling pathways in Bcr-Abl+ CML cells, which were previously thought to be activated directly by Bcr-Abl. 2) Although Jak2 is activated by Bcr-Abl, Jak2 is the dominant target molecule in CML and Jak2 becomes even more dominant in CML cells that become resistant to IM and other tyrosine kinase inhibitors. These findings indicate that CML cells, because of the dominant role of Jak2, resemble leukemia cells from Bcr-Abl negative myeloproliferative diseases. Disclosures: No relevant conflicts of interest to declare.

Effects of NVP-BSK805, a Novel JAK2 Inhibitor, on BCR-ABL and JAK2V617F Positive Cell Lines

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5002-5002
Author(s):  
Frauke Ringel ◽  
Jaspal S Kaeda ◽  
Michaela Schwarz ◽  
Peggy Grille ◽  
Bernd Dörken ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Cell Line ◽  
Cell Lines ◽  
Synergistic Effects ◽  
The Other ◽  
Jak2 Inhibitor ◽  
Ic50 Value ◽  
V617f Mutation ◽  
Jak2 Inhibition ◽  
Stat Pathway

Abstract Abstract 5002 Background: Janus kinases are critical components of cytokine signaling pathways that regulate hematopoiesis, growth, immunity, inflammation, and development. Oncogenic mutations of the non-receptor tyrosine kinase JAK2 are found in many Philadelphia chromosome negative myeloproliferative neoplasms. The V617F mutation in JAK2 occurs in 95% of patients with polycythemia vera, 50% of those with essential thrombocythemia and 50% of primary myelofibrosis patients. Preclinical results strongly support that JAK2 inhibitors could be effectively used in these three indications. Replacement of valine 617 with phenylalanine upregulates the tyrosine kinase activity of JAK2, causing constitutive activation of the JAK-STAT pathway and growth factor-independent cell proliferation. JAK2 has also been postulated to play an important role in BCR-ABL signal transduction. Therefore, inhibitors of the tyrosine kinase activity of JAK2 are under investigation as new therapy strategies for CMPNs. In this study the role of the novel JAK2 inhibitor, NVP-BSK805 (Novartis Pharmaceuticals), has been investigated in cells expressing either BCR-ABL or mutant JAK2. Possible synergistic effects between NVP-BSK805 and the already established tyrosine kinase inhibitors imatinib and nilotinib were assessed. Methods: The in vitro activity of NVP-BSK805 was analyzed in 12 hematopoietic cell lines, including 7 BCR-ABL positive (K562, KCL22, KU812, Lama87, BV173, EM3, SUP-B15), 4 JAK2 mutated (CHRF288, SET2, UKE1, HEL), the T-cell leukemia cell line Jurkat, and the neuroendocrine colonic tumour line LCC-18. Concentration kinetics from 0 up to 25 μM were established using XTT proliferation assays and flow cytometry for measuring apoptosis. Protein levels of JAK2, phospho-JAK2, STAT5, phospho-STAT5 and BCR-ABL were analyzed using Western blotting. NVP-BSK805 was also tested in combination with imatinib and nilotinib. JAK2 was sequenced in all cell lines in order to detect possible mutations in the gene. Results: Of the JAK2 mutated cell lines tested, 3 of 4 (CHRF288, SET2, UKE1) showed a significant reduction of proliferation, as well as viability, compared to the other cell lines. CHRF288 responded best to NVP-BSK805 with an IC50 value of 0.22 ± 0.04 μM. UKE1 and SET2 had similar values of 0.35 ± 0.03 μM and 0.37 ± 0.05 μM. Interestingly, HEL (V617F positive) cells showed only an IC50 value (1.8 ± 0.17 μM) for NVP-BSK805, comparable with that of the non-mutated BCR-ABL positive cell lines (1.5 to 2.7 μM). LCC-18 showed the weakest response of all cell lines tested, with an IC50 value of 9.93 ± 0.202 μM. Each cell line responded to concentrations higher than 5 μM with a strong reduction of proliferation due to inhibition of various kinases. Combination of the JAK2 inhibitor with imatinib and nilotinib showed no significant additive or synergistic effects, although all BCR-ABL positive cell lines responded well to both CML therapeutic agents. Western blotting of proteins of the JAK-STAT pathway confirmed the results of the proliferation and apoptosis tests showing a strong reduction of phoshorylated STAT5 in CHRF288 cells after a 30 min incubation even with NVP-BSK805 concentrations as low as 0.01 μM. UKE-1 and SET-2 showed reduction of pSTAT5 from 0.1 μM. Levels of total STAT5 were not affected. In all the other cell lines no changes were detected in any of the proteins tested. Conclusions: Here, we tested a novel JAK2 inhibitor in cells carrying the V617F mutation. Interestingly, not every cell line with the JAK2 V617F mutation showed a good response upon JAK2 inhibition, indicating that there are additional factors determining response. On the other hand, clinical trials with JAK inhibitors in myelofibrosis have shown responses in V617F-mutated and non-mutated patients, warranting further research to identify predictors of response. In BCR-ABL mutant cells not harbouring JAK2 mutations no significant inhibition of proliferation or apoptosis was detected following JAK2 inhibition, indicating that there are JAK2 independent signal transduction pathways of BCR-ABL to avoid apoptosis. Disclosures: le Coutre: Novartis Pharmaceuticals: Honoraria, Research Funding.

scholarly journals DDRE-03. IDH1-MUTANT GBM CELLS ARE HIGHLY SENSITIVE TO COMBINATION OF KDM6A/B AND HDAC INHIBITORS

2020 ◽  
Vol 3 (Supplement_1) ◽  
pp. i6-i7
Author(s):  
Alişan Kayabölen ◽  
Gizem Nur Sahin ◽  
Fidan Seker ◽  
Ahmet Cingöz ◽  
Bekir Isik ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Mutant Cell ◽  
Glioma Cells ◽  
Epigenetic Therapy ◽  
Low Grade ◽  
Orthotopic Model ◽  
Cycle Arrest ◽  
Mutant Cells

Abstract Mutations in IDH1 and IDH2 genes are common in low grade gliomas and secondary GBM and are known to cause a distinct epigenetic landscape in these tumors. To interrogate the epigenetic vulnerabilities of IDH-mutant gliomas, we performed a chemical screen with inhibitors of chromatin modifiers and identified 5-azacytidine, Chaetocin, GSK-J4 and Belinostat as potent agents against primary IDH1-mutant cell lines. Testing the combinatorial efficacy of these agents, we demonstrated GSK-J4 and Belinostat combination as a very effective treatment for the IDH1-mutant glioma cells. Engineering established cell lines to ectopically express IDH1R132H, we showed that IDH1R132H cells adopted a different transcriptome with changes in stress-related pathways that were reversible with the mutant IDH1 inhibitor, GSK864. The combination of GSK-J4 and Belinostat was highly effective on IDH1R132H cells, but not on wt glioma cells or nonmalignant fibroblasts and astrocytes. The cell death induced by GSK-J4 and Belinostat combination involved the induction of cell cycle arrest and apoptosis. RNA sequencing analyses revealed activation of inflammatory and unfolded protein response pathways in IDH1-mutant cells upon treatment with GSK-J4 and Belinostat conferring increased stress to glioma cells. Specifically, GSK-J4 induced ATF4-mediated integrated stress response and Belinostat induced cell cycle arrest in primary IDH1-mutant glioma cells; which were accompanied by DDIT3/CHOP-dependent upregulation of apoptosis. Moreover, to dissect out the responsible target histone demethylase, we undertook genetic approach and demonstrated that CRISPR/Cas9 mediated ablation of both KDM6A and KDM6B genes phenocopied the effects of GSK-J4 in IDH1-mutant cells. Finally, GSK-J4 and Belinostat combination significantly decreased tumor growth and increased survival in an orthotopic model in mice. Together, these results suggest a potential combination epigenetic therapy against IDH1-mutant gliomas.

scholarly journals Ruxolitinib Combined with Gemcitabine against Cholangiocarcinoma Growth via the JAK2/STAT1/3/ALDH1A3 Pathway

Biomedicines ◽  
2021 ◽  
Vol 9 (8) ◽  
pp. 885
Author(s):  
Shin-Yi Chung ◽  
Yi-Ping Hung ◽  
Yi-Ru Pan ◽  
Yu-Chan Chang ◽  
Chiao-En Wu ◽  
...  
Keyword(s):  
Cell Lines ◽  
Nuclear Translocation ◽  
Vital Role ◽  
First Line ◽  
Current Standard ◽  
Integrated Network ◽  
Jak2 Inhibitor ◽  
Therapeutic Drugs ◽  
Cholangiocarcinoma Cell ◽  
Malignant Behavior

Cholangiocarcinoma is the most common primary malignant tumor of the bile duct. The current standard first-line treatment for advanced or metastatic cholangiocarcinoma is gemcitabine and cisplatin. However, few effective treatment choices exist for refractory cholangiocarcinoma, and additional therapeutic drugs are urgently required. Our previous work demonstrated that the ALDH isoform 1A3 plays a vital role in the malignant behavior of cholangiocarcinoma and may serve as a new therapeutic target. In this study, we found a positive correlation between ALDH1A3 protein expression levels and the cell migration abilities of three cholangiocarcinoma cell lines, which was verified using ALDH1A3-overexpressing and ALDH1A3-knockdown clones. We also used ALDH1A3-high and ALDH1A3-low populations of cholangiocarcinoma cell lines from the library of integrated network-based cellular signatures (LINCS) program and assessed the effects of ruxolitinib, a commercially available JAK2 inhibitor. Ruxolitinib had a higher cytotoxic effect when combined with gemcitabine. Furthermore, the nuclear translocation STAT1 and STAT3 heterodimers were markedly diminished by ruxolitinib treatment, possibly resulting in decreased ALDH1A3 activation. Notably, ruxolitinib alone or combined with gemcitabine led to significantly reduced tumor size and weight. Collectively, our studies suggest that ruxolitinib might suppress the ALDH1A3 activation through the JAK2/STAT1/3 pathway in cholangiocarcinoma, and trials should be undertaken to evaluate its efficacy in clinical therapy.

scholarly journals Dengue Virus Induces the Expression and Release of Endocan from Endothelial Cells by an NS1–TLR4-Dependent Mechanism

2021 ◽  
Vol 9 (6) ◽  
pp. 1305
Author(s):  
Carlos Alonso Domínguez-Alemán ◽  
Luis Alberto Sánchez-Vargas ◽  
Karina Guadalupe Hernández-Flores ◽  
Andrea Isabel Torres-Zugaide ◽  
Arturo Reyes-Sandoval ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Mrna Expression ◽  
Cell Lines ◽  
Cell Activation ◽  
Dengue Infection ◽  
Elevated Serum ◽  
Fold Increase ◽  
Endothelial Cell Activation ◽  
Stimulation Of

A common hallmark of dengue infections is the dysfunction of the vascular endothelium induced by different biological mechanisms. In this paper, we studied the role of recombinant NS1 proteins representing the four dengue serotypes, and their role in promoting the expression and release of endocan, which is a highly specific biomarker of endothelial cell activation. We evaluated mRNA expression and the levels of endocan protein in vitro following the stimulation of HUVEC and HMEC-1 cell lines with recombinant NS1 proteins. NS1 proteins increase endocan mRNA expression 48 h post-activation in both endothelial cell lines. Endocan mRNA expression levels were higher in HUVEC and HMEC-1 cells stimulated with NS1 proteins than in non-stimulated cells (p < 0.05). A two-fold to three-fold increase in endocan protein release was observed after the stimulation of HUVECs or HMEC-1 cells with NS1 proteins compared with that in non-stimulated cells (p < 0.05). The blockade of Toll-like receptor 4 (TLR-4) signaling on HMEC-1 cells with an antagonistic antibody prevented NS1-dependent endocan production. Dengue-infected patients showed elevated serum endocan levels (≥30 ng/mL) during early dengue infection. High endocan serum levels were associated with laboratory abnormalities, such as lymphopenia and thrombocytopenia, and are associated with the presence of NS1 in the serum.

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