Acquired Resistance to JAK Inhibitors in Calr-Mutated Myeloproliferative Neoplasms

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.

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Oxidative Stress Induces Binding of FANCD2 to STAT5 and Facilitates STAT5-Dependent Survival Signals.

Blood ◽

10.1182/blood.v104.11.33.33 ◽

2004 ◽

Vol 104 (11) ◽

pp. 33-33 ◽

Cited By ~ 1

Author(s):

Grover Bagby ◽

Winifred Keeble ◽

Tara Koretsky ◽

Dylan Zodrow ◽

Richard Jove ◽

...

Keyword(s):

Oxidative Stress ◽

Ground State ◽

Mutant Cell ◽

Extracellular Proteins ◽

Wild Type ◽

Direct Role ◽

Normal Cells ◽

Jak2 Inhibitor ◽

Stat Signaling ◽

Mutant Cells

Abstract Fanconi anemia (FA) cells are hypersensitive to oxidative stress and exhibit aberrant STAT activation responses to defined extracellular proteins but whether these abnormalities are linked is unclear. Because oxidative stress is known to induce STAT activation, we hypothesized that proper STAT signaling responses in normal cells exposed to H2O2 require intact FA proteins. In fact, we found that FA-C, FA-G, and FA-D2 cells (fibroblasts) showed a significant increase in apoptosis after H2O2-exposure compared to retrovirally-complemented cells. H2O2 induced higher phospho-STAT5 (P-STAT5) expression in complemented cells than in mutant cells. Conversely, mutant cells expressed higher levels of P-STAT3 in both the ground state and after H2O2-induction than complemented cells. Aberrant STAT activation in FA mutant cells was shown to be both nucleus- and JAK2 kinase-dependent. Only low levels of STAT3 and STAT5 were induced in both mutant and complemented cytoplasts and AG490 (a Jak2 inhibitor) significantly suppressed H2O2-induced STAT5 responses. Seeking a direct role of FANCD2 in regulating proper STAT activation responses to H2O2, we carried out immunoprecipitation experiments (with an antibody to the N-terminal fragment of FANCD2) using PD20, a FA-D2 mutant cell line, and FANCD2 complemented PD20. In FANCD2-complemented and normal cells, anti-FANCD2 antibody immunoprecipitated STAT5. However, in mutant cells the same antibody immunoprecipitated STAT3, not STAT5. Thus, mutant (truncated) FANCD2 preferentially binds to and may activate STAT3 in the ground state. In fact, wild type FANCD2 also binds aberrantly to STAT3 in HSC536 (FA-C lymphoblasts) indicating that FANCC may influence the function of wild type FANCD2 and that binding of wild type FANCD2 to STAT3 does not require FANCD2 ubiquitinylation (FANCD2 is not ubiquitinylated in FA-C). Suspecting that in H2O2-exposed cells STAT5 signaling pathways lead to survival while STAT3 pathways lead to apoptosis, we transduced constitutively active mutants (*) of STATs 3 and 5 in mutant D2 and complemented cells. STAT3* increased apoptotic responses to H2O2 in complemented FA-D2 cells and STAT5* decreased apoptotic responses in H2O2-induced FA-D2 cells. In addition, the STAT5 inducible anti-apoptotic gene Bcl-XL was induced in H2O2-exposed complemented FA-D2 cells but not in FA-D2 cells. We conclude that FANCD2 functions to promote survival by ordering proper STAT signaling responses to oxidative stress and that this function of FANCD2 depends in part upon FA-C. We propose that FA cells are hypersensitive to oxidative stress in part because of imbalanced STAT signal transduction responses.

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Type II Inhibition of JAK2 with NVP-CHZ868 Reverses Type I JAK Inhibitor Persistence and Demonstrates Increased Efficacy in MPN Models

Blood ◽

10.1182/blood.v124.21.160.160 ◽

2014 ◽

Vol 124 (21) ◽

pp. 160-160 ◽

Cited By ~ 1

Author(s):

Sara C Meyer ◽

Matthew D Keller ◽

Priya Koppikar ◽

Olga A Guryanova ◽

Maria Kleppe ◽

...

Keyword(s):

Bone Marrow ◽

Clinical Development ◽

Type I ◽

Significant Activity ◽

Type Ii ◽

Jak Inhibitor ◽

Jak Inhibitors ◽

Allele Burden ◽

Stat Signaling ◽

Mutant Cells

Abstract The identification of JAK2 mutations in patients with myeloproliferative neoplasms (MPN) led to the clinical development of JAK2 inhibitors, and the JAK1/2 inhibitor ruxolitinib has been approved for the treatment of myelofibrosis (MF). Although clinically tested JAK inhibitors improve MPN-associated splenomegaly and systemic symptoms, they do not significantly reduce the MPN clone in most MPN patients.We previously demonstrated that MPN cells can acquire persistence to ruxolitinib and other type I JAK inhibitors which bind the active conformation of JAK2, and that JAK2 inhibitor persistence is associated with reactivation of JAK-STAT signaling and with heterodimerization between activated JAK2 and JAK1/TYK2, consistent with activation of JAK2 in trans by other JAK kinases. We have now extended our studies to other type I JAK inhibitors in clinical development, including CYT387, BMS911543 and SAR302503. In each case we see the same mechanism of persistence as observed with ruxolitinib, with transactivation of JAK2 by other JAK kinases. Most importantly, we found that MPN cells which were persistent to one JAK inhibitor were insensitive to the other JAK inhibitors, suggesting that the mechanisms which limit overall efficacy of ruxolitinib will limit the efficacy of other JAK inhibitors in clinical development. All JAK inhibitors in clinical development are type I inhibitors that interact with and inhibit the active confirmation of the JAK2 kinase. We hypothesized that novel, type II JAK inhibitors that interact with and inhibit JAK2 in the inactive conformation might retain activity in JAK inhibitor persistent cells and show increased efficacy in murine MPN models. We therefore characterized the efficacy of NVP-CHZ868, a novel type II JAK inhibitor, in MPN cells and in murine MPN models. CHZ868 potently inhibited proliferation of cells expressing the JAK2V617F mutation or the TEL-JAK2 fusion. We found that JAK2/MPL-mutant cell lines were universally sensitive to NVP-CHZ868. CHZ868 treatment of JAK2-mutant SET2 cells induced a higher degree of apoptosis compared to ruxolitinib. Signaling studies demonstrated that CHZ868 more potently attenuated JAK-STAT signaling in JAK2/MPL-mutant cells, with suppression of JAK2 phosphorylation consistent with a type II mechanism of kinase inhibition. We next investigated the ability of CHZ868 to inhibit the proliferation and signaling of MPN cells that had acquired persistence to type I JAK inhibitors. Type II inhibition with CHZ868 completely suppressed JAK-STAT signaling in type I JAK inhibitor-persistent cells, and prevented heterodimeric activation of JAK2 by JAK1 and TYK2. Most importantly, JAK2/MPL-mutant cells which were insensitive to type I JAK inhibitors remained highly sensitive to CHZ868, demonstrating that type I JAK inhibitor persistence does not confer resistance to type II inhibitors. We next evaluated the efficacy of CHZ868 in murine models of JAK2/MPL-mutant MPN. CHZ868 showed significant activity in conditional knock-in and bone marrow transplant (BMT) models of Jak2V617F-induced polycythemia vera, with normalization of hematocrit, reversal of stem/progenitor expansion, normalization of splenomegaly/splenic architecture, and reversal of bone marrow fibrosis. CHZ868 demonstrated similar activity in the MPLW515L BMT model of MF, with normalization of blood counts, stem/progenitor expansion, spleen weights, and extramedullary hematopoiesis in vivo. Most importantly, CHZ868 resulted in significant reductions of mutant allele burden (mean allele burden reduction 49%) in the Jak2V617F model. We observed analogous reductions in allele burden in the Jak2V617F and MPLW515L BMT models, consistent with disease modifying activity. Taken together, our data demonstrate that a spectrum of type I JAK inhibitors induce JAK inhibitor persistence, by a similar mechanism of JAK2 transactivation as observed with ruxolitinib. By contrast, type II JAK inhibition with CHZ868 remains highly active in JAK inhibitor persistent cells, and shows increased activity in murine MPN models. These data demonstrate that novel JAK inhibitors can increase target inhibition and therapeutic efficacy and should be pursued as an approach to improve outcomes for MPN patients. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Koppikar: Amgen: Employment. Sellers:Novartis: Employment. Hofmann:Novartis: Employment. Baffert:Novartis: Employment. Gaul:Novartis: Employment. Radimerski:Novartis: Employment. Levine:Novartis: Consultancy, Grant support Other.

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

Blood ◽

10.1182/blood.v118.21.122.122 ◽

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.

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The Novel JAK2 Inhibitor NVP-BSK805 Has Cytotoxic Activity on Malignant Plasma Cells

Blood ◽

10.1182/blood.v116.21.2993.2993 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2993-2993

Author(s):

Renate Burger ◽

Franziska Rademacher ◽

Matthias Staudinger ◽

Matthias Peipp ◽

Andreas Güunther ◽

...

Keyword(s):

Cytotoxic Activity ◽

Cell Lines ◽

Plasma Cell ◽

Stromal Cells ◽

Plasma Cells ◽

Myeloma Cell ◽

Synergistic Activity ◽

Jak Inhibitors ◽

Inhibitory Effects ◽

Jak2 Inhibitor

Abstract Abstract 2993 In multiple myeloma (MM) and plasma cell leukemia, activation of the JAK/STAT pathway is induced by interleukin (IL)-6, which is produced and secreted into the tumor microenvironment primarily by stromal cells. Upon binding of IL-6 to its specific alpha-chain receptor, dimerization of the gp130 signaling subunits leads to activation of associated JAK kinases and STAT transcription factors. In particular, STAT3 has been shown to be essential for myeloma cell growth and survival. NVP-BSK805 (Novartis) is a novel substituted quinoxaline JAK2 inhibitor tool compound which displays more than 20-fold selectivity for JAK2 over the other JAK family members and more than 100-fold selectivity over a panel of additional kinases (Baffert et al., Mol Cancer Ther 9:1945, 2010). The study presented here aims at growth inhibitory effects of NVP-BSK805 in malignant plasma cells. NVP-BSK805 inhibited the growth of six human myeloma cell lines displaying dose-dependent activity with IC50 concentrations between 2.6 μ mol/L and 6.8 μ mol/L. Among the cell lines, IL-6 dependent INA-6 cells were most sensitive to the inhibitory effects of the compound: both IL-6 and bone marrow stromal cell induced proliferation as measured by [3H]-thymidine uptake was completely inhibited at 4 μ mol/L and IC50 concentrations were less than 1 μ mol/L. Viability of the stromal cells was not significantly affected. NVP-BSK805 concentrations as low as 0.5 μ mol/L were sufficient to yield a marked reduction of IL-6 induced STAT3 phosphorylation and complete abrogation at 2 μ mol/L, thereby blocking essential survival signals. Accordingly, treatment of INA-6 cells with NVP-BSK805 for 48 hours led to significant apoptosis starting at 2 μ mol/L with a 30% increase in annexin V-positive cell numbers compared to DMSO controls. Importantly, NVP-BSK805 showed potent cytotoxic activity on plasma cell-enriched primary tumor samples from patients with extramedullary plasma cell disease that are highly responsive to IL-6: in 3 out of 4 tumor samples the IC50 concentrations were between 0.5 μ mol/L and 0.6 μ mol/L. These studies are extended to combinations of NVP-BSK805 with PI3K, mToR, MAPK, HDAC, and IGF-1R inhibitors in order to optimize targeted therapy strategies facing different pathway alterations in individual myeloma patients. In INA-6 cells, synergistic activity was found combining NVP-BSK805 with rapamycin and the MEK1 inhibitor U0126. Preclinical in vivo studies are ongoing. Our results with NVP-BSK805 substantiate the use of JAK inhibitors as a therapeutic strategy for patients with MM. Since results from studies with pan-JAK inhibitors such as pyridone 6 indicate involvement of additional JAK kinases, the choice of the optimal compound will depend on its JAK family selectivity and the biology of JAK signaling in MM. Disclosures: No relevant conflicts of interest to declare.

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Interferon-γ-Induced Upregulation of Immunoproteasome Subunit Assembly Overcomes Bortezomib Resistance of Leukemia Cell Lines Harbouring Bortezomib-Induced Mutations in Constitutive PSMB5

Blood ◽

10.1182/blood.v120.21.1346.1346 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1346-1346 ◽

Cited By ~ 1

Author(s):

Denise Niewerth ◽

Niels Franke ◽

Gerrit Jansen ◽

Yehuda Assaraf ◽

Johan van Meerloo ◽

...

Keyword(s):

Cell Lines ◽

Acquired Resistance ◽

Hematologic Malignancies ◽

Interferon Γ ◽

Resistant Cell ◽

Proteasome Activity ◽

Mrna Levels ◽

Wild Type ◽

Ic50 Values ◽

Ifn Γ

Abstract Abstract 1346 Acquired resistance to the proteasome inhibitor (PI) bortezomib (BTZ) is an emerging factor limiting its efficacy in the treatment of hematologic malignancies. The clinical impact of acquired resistance has been shown in Multiple Myeloma (MM) patients who were re-treated with BTZ. Although BTZ-retreatment was found to be effective, the response rate as well as the duration of response were less as compared to initial treatment, indicating the development of BTZ-resistance in a subgroup of patients. In line with that, we previously found increased expression of constitutive proteasome (cP) subunit ß5 harbouring a mutation in the BTZ-binding pocket and a decreased expression of non-mutated immunoproteasome subunits in BTZ-resistant cell lines of hematologic malignancies (Franke and Niewerth et al, Leukemia 2012). We here explore whether upregulation of immunoproteasome (iP) expression could restore sensitivity in BTZ-resistant leukemia cells towards BTZ and two epxoyketone-based irreversible PIs; carfilzomib (CFZ) and the ß5i-targeted ONX 0914. BTZ-resistant cell lines were of multiple myeloma (8226), T-cell (CEM) and myelomonocytic (THP1) origin and displayed resistance towards cell growth inhibition in the presence of 7–200 nM BTZ. Induction of iP in wild type (WT) and BTZ-resistant 8226, CCRF-CEM and THP1 cells was achieved by exposure to 100U/ml Interferon- γ (IFN-γ) for 6–72 h. IFN-γ transiently increased (maximum between 24–48 hours) mRNA levels of β5i, β1i, and β2i up to 8-fold, 30-fold and 4-fold, respectively. These findings were corroborated at the β5i, β1i and β2i protein expression level using Western blot analysis. Following IFN-γ exposure, chymotrypsin-like proteasome activity increased up to 2.5-fold compared to unstimulated controls, trypsin-like activity increased up to 1.5-fold, whereas caspase-like activity was slightly decreased. Consistent with increased proteasome activity, there was also an increased expression of cell surface HLA Class I molecules. The impact of IFN-γ induced upregulation of iPs on the sensitivity to the PI BTZ, CFZ, and ONX 0914, defined by the decrease in IC50, is summarized in Table 1. 8226/BTZ100 cells became 4-fold more sensitive towards BTZ after IFN-γ exposure, whereas THP1/BTZ200 and CEM/BTZ200 cells displayed nearly 2-fold increased sensitivity. For CFZ, a modest level of sensitization was observed in all cell lines with high level BTZ resistance. Interestingly, for the immunoproteasome inhibitor ONX 0914, IC50 values were markedly decreased (7-fold for 8226/BTZ100 and 3-fold for THP1/BTZ200 and CEM/BTZ200 cells). Additionally, in 8226 cells with low levels of BTZ resistance (8226/BTZ7), IFN-γ restored parental cell sensitivity to ONX 0914. Restoration of PI sensitivity after IFN-γ exposure was further confirmed by activation of PARP cleavage and accumulation of ubiquitinated proteins, pointing to restoration of BTZ activity under proteasome inhibition and consequent induction of apoptosis. Finally, to provide evidence that upregulation of β5i and or β1i by IFN-γ was responsible for the observed sensitization, siRNA downregulation of β5i and β1i was applied prior to exposure to IFN-γ. Under these conditions, mRNA levels and proteasome activity of β5i remained suppressed, even after exposure to IFN-γ. Moreover, after β5i silencing, PI sensitization and apoptosis were attenuated. Silencing of β1i expression had no effect on PI-sensitization. In conclusion, down-regulation of β5i subunit expression is a major determinant of BTZ-resistance and increasing its proteasomal assembly after IFN-γ exposure facilitates restoration of sensitivity in BTZ-resistant leukemia cells towards cP inhibitors and in particular iP inhibitors. Table 1. IC50 values of PIs ± IFN-γ pre-incubation (48 hr) of wild type and BTZ-resistant hematologic cell lines Cell lines BTZ BTZ + IFNy SF ONX 0914 ONX 0914 + IFNy SF CFZ CFZ + IFNy SF 8226/wt 2.6 1.8 1.4 54 46 1.5 0.4 0.4 1 8226/BTZ7 13.5 5.8 2.3 99 47 2.1 0.9 0.8 1.1 8226/BTZ100 208 57 3.6 1837 249 7.4 28 13 2.2 CEM/wt 4.1 3.9 1.1 75 65 1.2 0.4 0.3 1.3 CEM/BTZ200 416 223 1.9 1763 566 3.1 42 26 1.6 THP1/wt 6.2 5.1 1.2 52 19 2.7 0.9 1.3 0.7 THP1/BTZ200 641 347 1.8 4236 1376 3.1 49 34 1.4 50% inhibitory concentration compared to untreated controls (IC50 nM) as determined in a 4 days growth inhibition assay (MTT). Results depicted are means of at least 3 separate experiments. SF: sensitization factor: IC50 control/IC50 with IFN-g. Disclosures: No relevant conflicts of interest to declare.

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8-Azaguanine-resistant variants of cultured cells of Haplopappus gracilis

Canadian Journal of Botany ◽

10.1139/b78-320 ◽

1978 ◽

Vol 56 (21) ◽

pp. 2660-2665 ◽

Cited By ~ 6

Author(s):

Robert B. Horsch ◽

Gary E. Jones

Keyword(s):

Cell Lines ◽

Cultured Cells ◽

Wild Type ◽

Resistance Phenotype ◽

Cultured Cell ◽

Haplopappus Gracilis ◽

Purine Analog ◽

Mutant Cells ◽

Resistant Cells

Cultured cell lines of Haplopappus gracilis resistant to the purine analog 8-azaguanine (AG) have been isolated. One such line is 50 times more resistant to the drug than are wild-type cells. This line of AG-resistant cells also exhibits increased resistance to 8-azaadenine, but it responds to kinetin in a manner identical with that of wild-type cells. The resistance phenotype is stable for many generations in the absence of selection. In cultures of wild-type or mutant cells, more than 95% of cells are diploid.

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Hair follicle regeneration suppresses Ras-driven oncogenic growth

The Journal of Cell Biology ◽

10.1083/jcb.201907178 ◽

2019 ◽

Vol 218 (10) ◽

pp. 3212-3222 ◽

Cited By ~ 6

Author(s):

Cristiana M. Pineda ◽

David G. Gonzalez ◽

Catherine Matte-Martone ◽

Jonathan Boucher ◽

Elizabeth Lathrop ◽

...

Keyword(s):

Hair Follicle ◽

Normal Skin ◽

Tumor Development ◽

Hair Follicles ◽

Secondary Mutation ◽

Wild Type ◽

Clinically Normal ◽

Hair Follicle Stem Cells ◽

Follicle Stem Cells ◽

Mutant Cells

Mutations associated with tumor development in certain tissues can be nontumorigenic in others, yet the mechanisms underlying these different outcomes remains poorly understood. To address this, we targeted an activating Hras mutation to hair follicle stem cells and discovered that Hras mutant cells outcompete wild-type neighbors yet are integrated into clinically normal skin hair follicles. In contrast, targeting the Hras mutation to the upper noncycling region of the skin epithelium leads to benign outgrowths. Follicular Hras mutant cells autonomously and nonautonomously enhance regeneration, which directs mutant cells into continuous tissue cycling to promote integration rather than aberrancy. This follicular tolerance is maintained under additional challenges that promote tumorigenesis in the epidermis, including aging, injury, and a secondary mutation. Thus, the hair follicle possesses a unique, enhanced capacity to integrate and contain Hras mutant cells within both homeostatic and perturbed tissue, demonstrating that in the skin, multiple, distinct mechanisms exist to suppress oncogenic growth.

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Janus kinase inhibitors for the treatment of rheumatoid arthritis

Journal of Korean Medical Association ◽

10.5124/jkma.2021.64.2.105 ◽

2021 ◽

Vol 64 (2) ◽

pp. 105-108

Author(s):

Sun Hee Jang ◽

Ji Hyeon Ju

Keyword(s):

Rheumatoid Arthritis ◽

Kinase Inhibitors ◽

Interleukin 1 ◽

Janus Kinase ◽

Jak Inhibitor ◽

Jak Inhibitors ◽

Biologic Dmards ◽

Signal Transducers ◽

Refractory Rheumatoid Arthritis ◽

Stat Signaling

Rheumatoid arthritis is a chronic inflammatory destructive disorder that affects the joints, muscles, and tendons accompanying various extra-articular manifestations. Traditional disease-modifying anti-rheumatic drugs (DMARDs) represent the basic treatment for rheumatoid arthritis. Over the last 20 years, biologic DMARDs (tumor necrosis factor inhibitors, interleukin-1 inhibitors, interleukin-6 inhibitors, T cell inhibitors, and B cell inhibitors) have been widely used as a novel class of DMARDs that have efficacy and efficiency. Discovery of the underlying pathogenesis of autoimmune disease enables us to develop new target therapies such as a Janus kinase (JAK) inhibitor. Activated JAK is known to activate signal transducers as well as activators of transcription (STAT) signaling. A JAK inhibitor is a type of medication that functions by inhibiting the JAK-STAT signaling pathway. In addition, it is easy to take a JAK inhibitor orally. In Korea, several JAK inhibitors have been approved. This review describes the types of JAK inhibitors, recommended doses, side effects, and updated European Alliance of Associations for Rheumatology guidelines. Clinicians should more often consider JAK inhibitors in the treatment of refractory rheumatoid arthritis in current rheumatology clinics

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Potency and Selectivity Assessment of Small Molecules Against Janus Kinase (JAK) 2: Widely Used AG490 Inhibitor Is Neither Potent Nor Selective for JAK2

Blood ◽

10.1182/blood.v118.21.4780.4780 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4780-4780 ◽

Cited By ~ 1

Author(s):

Angus Sinclair ◽

Ivonne Archibeque ◽

Jinghui Zhan ◽

Liqin Liu ◽

Renee Emkey ◽

...

Keyword(s):

Cell Line ◽

Small Molecule ◽

Family Members ◽

Janus Kinase ◽

Published Data ◽

Enzyme Assays ◽

Jak Inhibitor ◽

Jak Inhibitors ◽

Jak2 Inhibitor ◽

Dependent Cell

Abstract Abstract 4780 Janus kinases (JAK) are the most proximal signaling components of multiple cytokine receptors and have four family members JAK1, JAK2, JAK3 and TYK2. JAK2 is essential for the development of normal erythroid and myeloid lineages by mediating signaling though the erythropoietin receptor (EPOR), thrombopoietin receptor (TPOR) and the β-common chain of GM-CSF, IL-3 and IL-5. Recently, JAK2 has been the focus of considerable research due to the discovery that patients with myeloproliferative disorders (MPDs) essential thrombocythemia, polycythemia vera and myelofibrosis contain somatically derived inactivating mutations in the JAK2 pseudokinase repressor domain. The deregulated expansion of erythro/myeloid cells in MPDs is thought to be due to the sustained signaling though JAK2 and downstream STAT, PI3K and MAPK signaling pathways to enhance the proliferation, survival and differentiation of progenitor cells. As a consequence, the discovery and development of small molecule inhibitors for JAK2 has been a focus for potential therapeutic intervention and has provided tools to examine cytokine networks. In order to discover small molecule JAK2 inhibitors we evaluated a number of benchmark and commercially available inhibitors as well as new inhibitors we generated. Tyrphostin AG490 has been widely used in the literature as a “JAK2” inhibitor in EPOR signaling and MPD research. However, AG490 has also been reported as an inhibitor of JAK3, EGFR, HER2, guanylyl cyclase C and BCR-ABL. In JAK2 enzyme assays, our new JAK inhibitors AMG-Jak2-02 and AMG-Jak2-03 were found to have transit IC50 <0.005 μM. However, AG490 was considered inactive in JAK2 enzyme assays with an IC50 >125 μM. When profiled against other JAK family members in enzyme assays, AG490 was also considered inactive on JAK1 (IC50 >125 μM), JAK3 (IC50 >80 μM) and TYK2 (IC50 >80 μM) whereas the IC50 of AMG-Jak2-02 and AMG-Jak2-03 were between 0.02 – 1.1 μM in JAK enzyme assays. Due to the lack of inhibitory activity of AG490 in JAK family enzyme assays, we performed a broader kinase screen on AG490. AG490 was profiled at 1 μM against 48 kinases. To our surprise, it was inactive on all kinases tested (most potent was SGK1 with ∼20% inhibition). In a broader binding screen of 441 lipid and protein kinases, 25 μM of AG490 was considered active on 4 kinases: STK17A, STK17B, PDGFRA and PDGFRB with a >70% inhibition. AG490 was inactive on all JAK family members. To investigate the potential for AG490 to inhibit JAK2 in a cellular context we examined the phosphorylation (p) of downstream molecules STAT5, AKT and ERK1/2 in an EPO dependent cell line UT-7/EPO. UT-7/EPO cells were incubated with AG490 (dose range up to 100 μM) or other JAK inhibitors (doses up to 33 or 100 μM) and phosphorylation of downstream molecules was assessed using Western immunoblot analysis. The EPO-EPOR induced downstream generation of pSTAT5, pAKT and pERK1/2 was suppressed by pan-JAK inhibitor I (Calbiochem) with an IC50 ∼ 0.1 μM, by AMG-Jak2-02 with an IC50 ∼ 10 μM and by AMG-Jak2-03 with an IC50 ∼ 0.1 μM. However, AG490 at 100 μM was unable to suppress the EPO-EPOR induced generation of pERK1/2 or pAKT but had modest effects on suppressing the generation of pSTAT5 (IC50 between 50–100 μM). We also investigated the potential for AG490 to inhibit the viability of JAK2 dependent (UT-7/EPO) and JAK2-independent (γ2A JAK2 null) cells. Pan JAK inhibitor I (Calbiochem), AMG-Jak2-01 and AMG-Jak2-02 were > 10 fold more potent at reducing JAK2 dependent cell viability (UT-7/EPO cells) compared with the viability of JAK2 independent cell line (γ2A cells). However, AG490 was found to be equipotent at inhibiting the viability of JAK2 dependent and independent cell lines. Similar results were obtained when these studies were repeated multiple times using multiple lots of compound (confirmed to be structurally correct based on NMR analysis). Taken together, we have identified that the widely used “JAK2” inhibitor AG490 is neither potent nor selective for JAK2. Thus, published data generated with AG490 should be interpreted with caution. Careful validation of JAK2 compounds for future research and assay development purposes should be taken into consideration. Disclosures: Sinclair: Amgen: Employment, Stock and Options. Archibeque:Amgen: Employment, Stock and Options. Zhan:Amgen, Inc: Employment, Stock and Options. Liu:Amgen, Inc: Employment, Stock and Options. Emkey:Amgen, Inc: Employment, Stock and Options. Doherty:Amgen, Inc: Employment, Stock and Options. Begley:Amgen, Inc: Employment, Stock and Options.

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