Prolonged Exposure to FLT3 Inhibitors Leads to Resistance Via Activation of Parallel Signaling Pathways.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1380-1380
Author(s):  
Obdulio Piloto ◽  
Melissa Wright ◽  
Patrick Brown ◽  
Kyu-Tae Kim ◽  
Mark Levis ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Kinase Inhibitors ◽  
Prolonged Exposure ◽  
Cellular Transformation ◽  
Mapk Signaling ◽  
Resistant Cell ◽  
Continuous Treatment

Abstract A number of tyrosine kinase inhibitors (TKI) have been developed to treat a variety of malignancies. However, continuous treatment with TKIs may select for resistant clones as has been seen with Gleevec treatment of CML. To study resistance to TKIs targeting FLT3, a receptor tyrosine kinase that is frequently mutated in AML, we developed resistant human cell lines through prolonged co-culture with FLT3 TKIs. Both FLT3 TKI sensitive and resistant cell lines exhibit inhibition of FLT3 phosphorylation upon FLT3 TKI treatment. However, FLT3 TKI resistant cell lines and primary samples often show continued activation of downstream PI3K/Akt and/or Ras/MEK/MAPK signaling pathways as well as continued expression of genes involved in FLT3-mediated cellular transformation. Inhibition of these pathways restores partial sensitivity to FLT3 TKIs. Mutational screening of FLT3 TKI resistant cell lines and primary samples failed to reveal any mutations in FLT3 or in 100 kinases/phosphatases tested but did reveal activating N-Ras mutations that were not present in the parental FLT3 TKI sensitive cell line. Taken together, these data indicate that FLT3 TKI resistant cells most frequently become FLT3 independent due to activation of parallel signaling pathways that provide compensatory survival / proliferation signals when FLT3 is inhibited. IMC-EB10, an unconjugated monoclonal antibody against FLT3, is still cytotoxic to FLT3 TKI resistant clones in vivo. An approach combining FLT3 TKIs with anti-FLT3 antibodies may prove superior and result in reduced chances of developing resistance.

scholarly journals Prolonged exposure to FLT3 inhibitors leads to resistance via activation of parallel signaling pathways

Blood ◽  
2006 ◽  
Vol 109 (4) ◽  
pp. 1643-1652 ◽  
Author(s):  
Obdulio Piloto ◽  
Melissa Wright ◽  
Patrick Brown ◽  
Kyu-Tae Kim ◽  
Mark Levis ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Kinase Inhibitors ◽  
Prolonged Exposure ◽  
Cellular Transformation ◽  
Mapk Signaling ◽  
Resistant Cell ◽  
Myelogenous Leukemia ◽  
Continuous Treatment

Abstract Continuous treatment of malignancies with tyrosine kinase inhibitors (TKIs) may select for resistant clones (ie, imatinib mesylate). To study resistance to TKIs targeting FLT3, a receptor tyrosine kinase that is frequently mutated in acute myelogenous leukemia (AML), we developed resistant human cell lines through prolonged coculture with FLT3 TKIs. FLT3 TKI-resistant cell lines and primary samples still exhibit inhibition of FLT3 phosphorylation on FLT3 TKI treatment. However, FLT3 TKI-resistant cell lines and primary samples often show continued activation of downstream PI3K/Akt and/or Ras/MEK/MAPK signaling pathways as well as continued expression of genes involved in FLT3-mediated cellular transformation. Inhibition of these signaling pathways restores partial sensitivity to FLT3 TKIs. Mutational screening of FLT3 TKI-resistant cell lines revealed activating N-Ras mutations in 2 cell lines that were not present in the parental FLT3 TKI-sensitive cell line. Taken together, these data indicate that FLT3 TKI-resistant cells most frequently become FLT3 independent because of activation of parallel signaling pathways that provide compensatory survival/proliferation signals when FLT3 is inhibited. Anti-FLT3 mAb treatment was still cytotoxic to FLT3 TKI-resistant clones. An approach combining FLT3 TKIs with anti-FLT3 antibodies and/or inhibitors of important pathways downstream of FLT3 may reduce the chances of developing resistance.

Targeting Mcl-1 Enhances the Activity of Tyrosine Kinase Inhibitor Gilteritinib in FLT3 Mutated AML

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 30-31
Author(s):  
Bing Z Carter ◽  
Po Yee Mak ◽  
Vivian Ruvolo ◽  
Wenjing Tao ◽  
Paul Hughes ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Signaling Pathways ◽  
Progenitor Cells ◽  
Research Funding ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Mapk Signaling ◽  
Culture Conditions ◽  
Beta Catenin ◽  
Adaptive Resistance

Anti-apoptotic Bcl-2 proteins play critical roles in AML cell and AML stem/progenitor cell survival and drug resistance, hence are relevant therapeutic targets. Indeed, the combination of the selective Bcl-2 inhibitor venetoclax (VEN) with a hypomethylating agent elicits CR/CRi rates of > 65%, is well tolerated by elderly AML patients, and obtained FDA approval. However, despite of the major improvement in response rates, survival extension was limited and most patients ultimately relapsed largely due to the development of resistant disease. Molecular analysis of treated patients revealed that primary and adaptive resistance to VEN-based combinations was frequently characterized by acquisition or enrichment of clones activating signaling pathways such as FLT3 or RAS (DiNardo CD et al., Blood 2020). FLT3 is one of the most frequently mutated gene in AML, resulting in constitutive activation of FLT3 tyrosine kinase and its downstream signaling pathways such as RAS/MAPK, which can be targeted by FLT3 tyrosine kinase inhibitors (TKIs). However, patients treated with TKIs ultimately relapse and adapt to TKI therapy by reactivating the MAPK signaling pathway (Bruner JK et al., Cancer Res 2017), which is known to stabilize Mcl-1 levels. Furthermore, deregulated Mcl-1 expression was identified as a novel mechanism of primary TKI resistance in a subset of FLT3-ITD mutated AML patients (Breitenbuecher F et al., Blood 2009). Importantly, Mcl-1 expression can be induced by VEN treatment and is a major resistance factor to VEN (Pan R et al., Cancer Discover 2014; Carter BZ et al., ASH 2018). Hence, Mcl-1 inhibition may enhance the efficacy of TKIs in FLT3 mutated AML, targeting AML cells and stem/progenitor cells. To determine if targeting Mcl-1 enhances the activity of TKIs in FLT3 mutated AML, we treated MV4-11 and Molm13 cells with Mcl-1 inhibitor AMG176 and TKI gilteritinib (GIL) and observed synergism, as defined by combination index < 1 in both cells. Mechanistic studies demonstrated that GIL markedly decreased Mcl-1 and antagonized AMG176-induced Mcl-1 induction. GIL and its combination with AMG176 also decreased Bcl-xL. Although Bcl-2 protein levels were largely not changed in MV4-11 cells, we found both single treatment and the combination greatly decreased Bcl-2 associated athanogene (BAG) proteins BAG1, BAG3, and BAG4 at the RNA level, which needs to be confirmed at the protein level. The BAG proteins are a family of chaperone regulators and BAG1 was reported to bind and enhance the activity of multiple proteins known to support cells survival, including Bcl-2 (Takayama S et al., Cell 1995). Interestingly, GIL treatment greatly diminished the levels of beta-catenin and its target protein c-Myc, consistent with our previous report that FLT3 regulates beta-catenin signaling (Xiang et al., CCR, 2018). We have generated Mcl-1 overexpressing (OE) and VEN-resistance (VEN-R) MV4-11 and Molm13 cells. The Mcl-1 OE cells are highly resistant to VEN and the VEN-R cells expressed high levels of Mcl-1. Combined inhibition of AMG176 and GIL synergistically induced cell death in Mcl-1 OE and VEN-R resistant cells. Although the expression is low in AML cells we tested, BCL2A1 is also known as a resistant factor to VEN. We generated BCL2A1 OE MV4-11 and Molm13 cells and demonstrated that combined inhibition of FLT3 and Mcl-1 was highly effective in these cells as well. Western blot analysis revealed that GIL effectively decreased Mcl-1 in Mcl-1 OE and VEN-R and BCL2A1 in BCL2A1 OE MV4-11 cells. Next, we treated FLT3 mutated AML patient samples harboring both, ITD and D835 mutations, from 2 patients who had both failed VEN-based therapy and from 1 patient with ITD mutation, with AMG176 and GIL under MSC co-culture conditions. Synergy was observed in all samples in AML blasts and AML stem/progenitor cells. Collectively, our data demonstrate that targeting Mcl-1 enhances the activity of GIL in FLT3 mutated AML, including those resistant to/relapsed from VEN-based therapy, findings that may warrant clinical evaluation. Disclosures Carter: Syndax: Research Funding; Ascentage: Research Funding; AstraZeneca: Research Funding; Amgen: Research Funding. Hughes:Amgen: Current Employment. Chen:Amgen: Current Employment. Morrow:Amgen: Current Employment. Andreeff:Amgen: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy.

Alox-5 As a Potent Therapeutic Target on Overcoming TKI-Resistance in Chronic Myeloid Leukemia with T315I Mutation in Bcr-Abl

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4835-4835 ◽  
Author(s):  
Jishi Wang ◽  
Dan Ma ◽  
Ping Wang ◽  
Weibing Wu ◽  
Lu Cao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Kinase Inhibitors ◽  
Mrna Level ◽  
Resistant Cell ◽  
Tki Resistance ◽  
T315i Mutation ◽  
Pkc Β

Abstract Background&Significance: Chronic myeloid leukemia(CML) is a malignant disease of a primitive haematological cell, characterised by inappropriate expansion of myeloid cells. Although the disease is readily controlled by Tyrosine kinase inhibitors, approximately one third of patients will eventually fail treatment. And we believed it corresponds to insensitive leukemia stem cells(LSCs) with unresponsive genes to the kinase inhibitors in CML and mutation of Bcr-Abl. 5-lipoxygenase gene(Alox5) was proved as a novel therapeutic target in cancer stem cells of CML. It encodes a member of the lipoxygenase gene family and plays a role in the synthesis of leukotrienes from arachidonic acid. Without Alox5, Bcr-Abl fails to induce CML in mice due to the impairments of the functions of LSCs. However, recent report suggest that Alox5 regulation may not be important for the development of CML in human. Interestingly, we found specific upregulation of Alox5 in CML patients with strongly positive expression of p210 in mRNA level, including the patients primary diagnosed as CML and who suffered in relapse for TKI resistance. Therefore, we characterized the function and regulation of Alox5 in TKI-resistant CML. Results: Firstly, we verified the upregulation of Alox5 by real-time PCR on sorted human CML progenitor populations with strongly positive expression of Bcr-Abl(p210), but not on CML patients obtained remission after treatment of TKI. To evaluate the function and regulation of Alox5, we silenced Alox5 by siRNA and chemical inhibitior in human CML cell lines K562, its TKI-resistant cell lines K562R, murine CML cell lines BaF3wild, and its TKI-resistant cell line BaF3T315I. As a result, the apoptotic rate induced by Alox5 inhibition alone in K562 and BaF3wild cells was lower than by TKI. Conversely, blockage of Alox5 in BaF3T315I cells caused increasingly apoptotic rate, which was higher than imatinib treatment. To further study if Alox5 could play an important role in impairing leukemia stem cells in CML, we cultured LSCs(Lin-c-Kit+Sca-1+) derived from CML patients with Bcr-Abl-T315I mutation in the presence of Alox5 inhibitor or imatinib alone. For 24h treatment, obvious apoptosis was observed in cells cultured with Zileuton, a kind of Alox5 inhibitor, but not in cells cultured with imatinib. Therefore, apoptosis related genes were detected, significant downregulation of Bcl-2 was found compared to in LSCs without Alox5 knockouted. In addition, downregulation of Alox5 followed Bcr-Abl inhibition in CML, we hypothesised that Alox5 was a downstream of Bcr-Abl, genomic array was used to uncover the signaling pathway connected Alox5 with Bcr-Abl. The results shown us that PKC-β was significantly downregulated when Bcr-Abl was inactivated by TKI. Selectivly inhibited PKC-β could decreased Alox5 expression in mRNA level. As for this mechanism, we found p38/MAPK signaling pathway mediated regulation of Alox5 by PKC-β. Next, we evaluated the in vivo anti-CML effect of Alox5 inhibition with a xenograft mice model. Two weeks after the transplantation of human CML cells with mutant Bcr-Abl-T315I(n=12). NOD/SCID/IL2Rg-KO(KOG) mice were treated with normal saline, 10mg/kg Zileuton or imatinib intraperitoneally on everyday schedule. At first, we confirmed that Zileuton doesn't affect normal hematopoiesis(n=3), but eventually inhibite LSCs growth(n=3). Eight to ten weeks after the transplantation, the frequencies of human CD45+ CML cells and LSCs were significantly reduced by Zileuton treatment in bone marrow((BM) of the recipient mice compared with normal saline-treated control mice and imatinib treated mice, indicating that Alox5 inhibition can inhibit the survival of CML-T315I mutant cells and LSCs in vivo. Conclusion: Together, these results suggest that Alox5 would be a potent therapeutic target on overcoming TKI-resistance involved in Bcr-Abl-T315I mutation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Regulation of Protein Kinase B Tyrosine Phosphorylation by Thyroid-Specific Oncogenic RET/PTC Kinases

2005 ◽  
Vol 19 (11) ◽  
pp. 2748-2759 ◽  
Author(s):  
Hye Sook Jung ◽  
Dong Wook Kim ◽  
Young Suk Jo ◽  
Hyo Kyun Chung ◽  
Jung Hun Song ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Signaling Pathways ◽  
Tyrosine Phosphorylation ◽  
Protein Kinase B ◽  
Cellular Transformation ◽  
Forkhead Transcription Factor ◽  
Src Tyrosine Kinase

Abstract Papillary thyroid carcinoma (PTC) is a heterogenous disorder characterized by unique gene rearrangements and gene mutations that activate signaling pathways responsible for cellular transformation, survival, and antiapoptosis. Activation of protein kinase B (PKB) and its downstream signaling pathways appears to be an important event in thyroid tumorigenesis. In this study, we found that the thyroid-specific oncogenic RET/PTC tyrosine kinase is able to phosphorylate PKB in vitro and in vivo. RET/PTC-transfected cells showed tyrosine phosphorylation of endogenous and exogenous PKB, which was independent of phosphorylation of T308 and S473 regulated by the upstream kinases phosphoinositide-dependent kinase-1 and -2, respectively. The PKB Y315 residue, which is known to be phosphorylated by Src tyrosine kinase, was also a major site of phosphorylation by RET/PTC. RET/PTC-mediated tyrosine phosphorylation results in the activation of PKB kinase activity. The activation of PKB by RET/PTC blocked the activity of the forkhead transcription factor, FKHRL1, but a Y315F mutant of PKB failed to inhibit FKHRL1 activity. In summary, these observations suggest that RET/PTC is able to phosphorylate the Y315 residue of PKB, an event that results in maximal activation of PKB for RET/PTC-induced thyroid tumorigenesis.

Role of IGF-1R inhibition on an Src-family kinase bypass resistance pathway: A rational basis for cotargeting IGF-1R and Src kinase in pediatric sarcomas?

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 10025-10025
Author(s):  
Xiaolin Wan ◽  
Choh L Yeung ◽  
Christine Heske ◽  
Arnulfo Mendoza ◽  
Lee J. Helman
Keyword(s):  
Signaling Pathways ◽  
Cell Lines ◽  
Kinase Inhibitors ◽  
Src Kinase ◽  
Es Cell ◽  
Src Family Kinase ◽  
Pediatric Sarcomas

10025 Background: Dysregulation of IGF signaling plays a fundamental role in oncogenesis in pediatric sarcomas. We recently completed a Phase II study targeting the IGFI receptor signaling pathway in refractory Ewing’s and other sarcomas. We demonstrated an objective response rate of 16 percent, but most responses were transient lasting less than 18 weeks. The majority of patients, even those with initial responses, do not have long term benefit from IGFIR blockade, indicating the presence of an innately resistant tumor mass or the recruitment of compensatory pathways allowing for continued growth. To improve on these responses, we have been probing these tumors to identify other critical pathways that might allow combined targeting approaches. Methods: Multiple RMS and ES cell lines were treated with IGF1R kinase inhibitors and assayed for up-regulation of various signaling pathways. Combination treatment with IGF1R inhibitors and inhibitors of additional signaling pathways were then tested in vitro and in vivo using standard techniques. For in vivo xenograft studies, treatments began 11 days following orthotopic injection of tumor cells. Results: We have identified repid up-regulation of Src family kinase (SFK) signaling within 4 hours of IGF1R blockade in both RMS and ES cell lines. Of note, combined treatment with IGF1R Ab plus IGF1R kinase inhibitors most potently upregulated SFK signaling. Based on these findings, we tested combined IGF1R blockade with SFK inhibition using the commercially available drug, dasatinib. We show that dual blockade of IGF1R and SFK pathways were synergistic in vitro. Furthermore, in xenograft models of RMS, the combination IGF1R and SFK inhibition led to long-term disease free status for at least 90 days in some mice, never seen in our hands previously using these models. Conclusions: This work identified that IGF-1R inhibition induced activation of Src kinase that may act as a by-pass pathway. Synergistic activity of IGF-1R and SFK kinase inhibitors was observed in vitro and in vivo. Dual IGFI and SFK kinase inhibition may lead to improved therapeutic outcomes.

Activity of the Multi-Targeted Kinase Inhibitor, AT9283 on Imatinib-Resistant CML Models.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1104-1104 ◽  
Author(s):  
Ruriko Tanaka ◽  
Matthew S Squires ◽  
Shinya Kimura ◽  
Asumi Yokota ◽  
Kirsty Mallett ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Cell Lines ◽  
Kinase Inhibitors ◽  
Wild Type ◽  
Once Daily ◽  
Imatinib Resistant ◽  
Mutant Forms ◽  
In Vivo Effects

Abstract CML is caused by a consistent genetic abnormality, termed the Philadelphia chromosome, that results from a reciprocal (9;22) translocation leading to the expression of the BCR-ABL fusion protein. Although treatment has been revolutionized by the introduction of tyrosine kinase inhibitors which target Abl activity, reactivation of Abl signaling via several different point mutations remains problematic. In particular the mutation of Threonine 315 to Isoleucine (T315I) confers resistance to all existing therapies with tyrosine kinase inhibitors in the clinical settings. We describe the in vitro and in vivo effects of AT9283, a potent inhibitor of several protein kinases, including Abl kinase (wild type BCR-ABL and several of the drug resistant mutant variants that have arisen in clinical practice e.g. T315I), JAK2, JAK3 and Aurora kinases A and B, on imatinib-resistant CML cells including those harboring BCR-ABL (T315I). AT9283 has potent anti-proliferative activity in a panel of BaF3 and human cell lines expressing the BCR-ABL or its mutant forms. In BaF3 BCR-ABL wild-type and T315I mutant cells and K562 CML cells we observed inhibition of substrates of both BCR-ABL (STAT5) and Aurora B (Histone H3) at concentrations >300nM and <100nM, respectively, suggesting that AT9283 is capable of inhibiting Aurora and BCR-ABL simultaneously in these cell lines. The in vivo effects of AT9283 were examined in several mouse models engrafted either subcutaneously or intravenously with BaF3, human CML cell lines or primary CML patient samples expressing the BCR-ABL or its mutant forms. Specifically AT9283 prolonged the survival of mice engrafted intravenously with either BaF3 BCR-ABL T315I, or E255K cells when administered intraperitoneally twice daily at doses of either 6.25 or 10mg/kg or once daily at 15mg/kg when administered 5 days in every week repeated twice. Maximal survival advantage was conferred at either 10mg/kg twice daily or 15mg/kg once a day. Similar data were obtained in an intravenous model using primary CML cells taken from a patient harbouring the BCR-ABL E255K mutation. We also present data from ongoing studies showing increased survival rates in these in vivo model systems following multiple cycles of AT9283 administered on the 15mg/kg once daily schedule. These data together support further clinical investigation of AT9283 in patients with treatment resistant CML.

Efficacy of the Novel Non-Quinone Based HSP-90 Inhibitor PU-H71 in JAK2V617F and MPLW515L-Induced Murine Models of Myeloproliferative Neoplasms.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3898-3898
Author(s):  
Priya Koppikar ◽  
Sachie Marubayashi ◽  
Tony Taldone ◽  
Omar Abdel-Wahab ◽  
Nathan West ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Cell Lines ◽  
Myeloproliferative Neoplasms ◽  
Leukemia Cell ◽  
Mapk Signaling ◽  
Hsp90 Inhibition ◽  
Stat Signaling ◽  
Hsp 90 ◽  
Jak2 Inhibitors

Abstract Abstract 3898 Poster Board III-834 The discovery of mutations in the JAK-STAT signaling pathway in the majority of patients with myeloproliferative neoplasms (MPN) has led to the development of JAK2 kinase inhibitors for the treatment of these disorders. Although JAK2 inhibitors demonstrate efficacy in preclinical models and in early phase clinical trials, to date, JAK2 inhibitor treatment has not resulted in molecular responses or in improvements in blood counts. We therefore have investigated the effects of additional therapies that might provide benefit to patients with myeloproliferative disorders, including Hsp90 inhibition, which has been shown to abrogate oncogenic signaling pathways in other human malignancies. We analyzed the effect of PU-H71, a novel non-quinone based Hsp-90 inhibitor, in MPN cell lines, primary patient samples, and in animal models. PU-H71 treatment caused potent, dose-dependent inhibition of cell growth in isogenic cell lines expressing JAK2/MPL mutations, JAK2V617F-positive leukemia cell lines, and primary MPN patient samples, which was associated with induction of apoptotic cell death at clinically achievable concentrations. Further, we observed JAK2 degradation in cell lines and primary samples with PU-H71 treatment, and immunoprecipitation experiments documented association of JAK2 with HSP90 and with PU-H71, demonstrating that JAK2 is a client of the HSP90 chaperone complex. PU-H71 potently inhibited downstream signaling pathways, including STAT signaling, MAPK signaling, and AKT signaling in JAK2/MPL positive cell lines and primary samples. Most importantly, in vivo therapy with PU-H71 in mice expressing JAK2V617F or MPLW515L normalized peripheral blood counts, attenuated extramedullary hematopoiesis, and improved survival compared to vehicle treated mice. We observed reduction in total JAK2 expression in target organs from PUH-71 treated mice, and noted in vivo inhibition of signaling pathways in a manner analogous to in vitro studies. Taken together, these data indicate that Hsp90 inhibition, either alone or in combination with JAK2 inhibitors, may prove useful against human MPN. Disclosures: Levine: Novartis: Research Funding; TargeGen: Consultancy.

The Bruton's Tyrosine Kinase (BTK) Inhibitor PCI-32765 Inhibits Growth of ABC DLBCL Tumors In Vivo and in Vitro by Preventing Activation of Pro-Survival NF-κB pathways

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4969-4969 ◽  
Author(s):  
Sriram Balasubramanian ◽  
Richard Crowley ◽  
Mint Sirisawad ◽  
Patti Thiemann ◽  
Jun Chen ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
B Cell ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Active Site ◽  
Employment Equity ◽  
Equity Ownership ◽  
Nuclear Relocation

Abstract Abstract 4969 Bruton's tyrosine kinase (BTK), a Tec family kinase essential to signaling via the B-cell Receptor (BCR) has emerged as a very promising drug target in lymphoma. PCI-32765, a potent and selective covalently-bound small molecule inhibitor of BTK (Honigberg, Proc. Natl Acad. Sci. USA 107:13075, 2010), has demonstrated promising clinical activity in a number of Phase I and II clinical trials in patients with chronic lymphocytic leukemia (CLL) (Byrd et al., ASCO 2011 Annual Meeting Abstracts, JCO29 (15): 8012, 2010) and several subtypes of B-cell Non-Hodgkin's Lymphoma (NHL), including diffuse large B-cell lymphoma (DLBCL). Many cell lines derived from B-cell malignancies do not show in vitro growth inhibition by BTK inhibitors, perhaps because most established B-cell lines no longer depend on BCR stimulation for survival. However, some cell lines derived from the aggressive ABC (activated B-cell) subtype of DLBCL such as OCI-Ly10 and TMD8 are very sensitive to BTK inhibition by PCI-32765 (Davis et al, Nature463: 88, 2010). We show here that both of these cell lines are potently and irreversibly inhibited by PCI-32765 (EC50 continuous exposure < 10 nM and EC50 following a 1-hr pulse < 50 nM). Interestingly, 2 other ABC-DLBCL cell lines, OCI-Ly3 and HBL-1, in which the NF-κB pathway is activated by a mutation in CARD11 which is downstream of BTK, are not inhibited by PCI-32765. Both sensitive and resistant cells express Btk at similar levels, and the active site of BTK is fully occupied in the presence of low concentrations of the inhibitor as shown using a fluorescently-labeled affinity probe. We show that the sensitive TMD8 and OCI-Ly10 cells have chronically active BCR signaling to NF-κB which is dose-dependently inhibited by PCI-32765, as measured by comparing the phosphorylation and nuclear relocation of NF-κB p65 subunit. These cells also show inhibition of other major signaling pathways downstream of the BCR, including p-ERK, p-AKT, p-PLCγ and intracellular calcium flux, ultimately leading to NF-κB activation. The non-responsive OCI-Ly3 cells do not show inhibition of NF-κB p65 phosphorylation or nuclear relocation, although Btk is present and active, as shown by the probe assay and inhibition of p-AKT by PCI-32765. Furthermore, whole genome expression analysis reveals inhibition of mRNA expression of several NF-κB target genes in OCI-Ly10, particularly c-Myc, NF-κB subunits and several chemokines and cytokines, leading to downregulation of STAT3 signaling, NFAT signaling, cell cycle and cytokine-chemokine signaling pathways, none of which was observed in OCI-Ly3 cells. In vivo, PCI-32765 dose-dependently inhibited tumor growth in mice carrying OCI-Ly10 (and TMD8, ongoing) xenografts. Analysis of these tumors exhibited dose-dependent occupancy of the BTK active site as well as inhibition of signaling pathways. These results demonstrate the mechanistic basis of PCI-32765 inhibitory activity in ABC-DLBCL and support the ongoing clinical investigation of the therapeutic use of PCI-32765 in patients with this devastating disease. Disclosures: Balasubramanian: Pharmacyclics: Employment, Equity Ownership. Crowley:Pharmacyclics: Employment, Equity Ownership. Sirisawad:Pharmacyclics: Employment, Equity Ownership. Thiemann:Pharmacyclics: Employment, Equity Ownership. Chen:Pharmacyclics: Employment, Equity Ownership. Buggy:Pharmacyclics, Inc.: Employment, Equity Ownership.

Elucidation of PLK1 Linked Biomarkers in Oesophageal Cancer Cell Lines: A Step Towards Novel Signaling Pathways by p53 and PLK1- Linked Functions Crosstalk

2020 ◽  
Vol 27 ◽  
Author(s):  
Nousheen Bibi ◽  
Ted Hupp ◽  
Mohammad Amjad Kamal ◽  
Sajid Rashid
Keyword(s):  
Signaling Pathways ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Kinase Inhibitors ◽  
Mass Spectrometry Data ◽  
Motif Analysis ◽  
P53 Expression ◽  
Proteomics Approach ◽  
The Guardian

Background: Oesophgeal adenocarcinoma (OAC) is the most frequent cause of cancer death. POLO-like kinase 1 (PLK1) is overexpressed in broad spectrum of tumors and has prognostic value in many cancers including esophageal cancer, suggesting its potential as a therapeutic target. p53, the guardian of genome is the most important tumor suppressors that represses the promoter of PLK1, whereas tumor cells with inactive p53 are arrested in mitosis due to DNA damage. PLK1 expression has been linked to the elevated p53 expression and has been shown to act as a biomarker that predicts poor prognosis in OAC. Objective: The aim of the present study was identification of PLK1 associated phosphorylation targets in p53 mutant and p53normal cells to explore the downstream signaliging evets. Methods: Here we develop a proof-of-concept phospho-proteomics approach to identify possible biomarkers that can be used to identify mutant p53 or wild-type p53 pathways. We treated PLK1 asynchronously followed by mass spectrometry data analysis. Protein networking and motif analysis tools were used to identify the significant clusters and potential biomarkers. Results: We investigated approximately 1300 potential PLK1-dependent phosphopeptides by LC-MS/MS. In total, 2216 and 1155 high confidence phosphosites were identified in CP-A (p53+)and OE33 (p53-) cell lines owing to PLK1 inhibition. Further clustering and motif assessment uncovered many significant biomarkers with known and novel link to PLK1. Conclusion: Taken together, our study suggests that PLK1 may serve as a potential therapeutic target in human OAC. The data highlight the efficacy and specificity of small molecule PLK1 kinase inhibitors to identify novel signaling pathways in vivo.

scholarly journals Combination of BCR-ABL1 Tyrosine Kinase Inhibitors with a Small Molecule LIM kinase1/2 Inhibitor in BCR-ABL1 Positive Acute Lymphoblastic Leukemia (ALL)

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2705-2705
Author(s):  
Thorsten Braun ◽  
Jeannig Berrou ◽  
Renaud Prudent ◽  
Hanane Djamai ◽  
Melanie Dupont ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Tyrosine Kinase ◽  
Cell Lines ◽  
Small Molecule ◽  
Research Funding ◽  
Kinase Inhibitors ◽  
Healthy Donors ◽  
Cd34 Cells

Abstract Introduction: LIM kinases 1 and 2 (LIMK1/2) are downstream effectors at the crossroads of different signaling pathways implicated in the dynamics of the cytoskeleton via phosphorylation of cofilin family proteins, degradation of the matrix by phosphorylating MT1-MMP and control of the activity of Aurora kinase A. Recently, the oncogenic role of Rho kinases (ROCK) was identified to be constitutively activated by BCR-ABL1, FLT3-ITD and KIT in hematologic malignancies via PI3 kinase and Rho GTPase mediated phosphorylation. Upon activation, ROCK phosphorylates LIMK1/2 leading to inactivation of cofilin by its phosphorylation and polymerization of actin and microtubules and possibly to other biological effects mediated by LIMK1/2, not yet fully understood. Here, we demonstrate synergy of a LIMK1/2 inhibitor with BCR-ABL1 tyrosine kinase inhibitors (TKI) in vitro and in vivo in different models for BCR-ABL1 driven ALL. Materials and Methods: Expression of LIMK1/2 was determined by RT-qPCR and WB in cell lines. Phosphorylation of cofilin was detected by WB. A small molecule inhibitor of LIMK1/2 was tested alone and in combination with imatinib, dasatinib, nilotinib and ponatinib in BCR-ABL1 positive ALL cell lines TOM-1 and BV-173. Cell viability and IC50 was assessed by MTS assays after exposure to LIMK1/2 inhibitor for 72h. In combination experiments, compounds were added simultaneously and relative cell numbers were determined at 72h with MTS assays and combination index (CI) values were calculated according to the Chou-Talalay model. Cell-cycle distribution was determined by cytofluorometric analysis detecting nuclear propidium iodide (PI) DNA intercalation. Induction of apoptosis was evaluated by annexin-V exposure and PI incorporation at 72h with increasing doses of LIMK1/2 inhibitor. Peripheral blood (PB) nucleated cells from apharesis products of healthy donors obtained after informed consent according to Helsinki declaration were incubated with or without LIMK1/2 inhibitor for 72h, and then enriched for CD34+ cells by immuno-magnetic selection and seeded in triplicate in methylcellulose FCS and cytokines. In vivo experiments were performed in C57Bl/6 mice injected with BCR-ABL-induced B-ALL cells. These were obtained by transduction of CDKN2A-deficient B-cell progenitors with a retrovirus coding for BCR-ABL1 (P185) and GFP, followed by transplantation in sub-lethally-irradiated recipient C57Bl/6 mice. Mice were treated either with LIMK1/2 inhibitor, nilotinib or the combination of both and compared to untreated control mice. Results: Expression of the two isoforms LIMK1 and LIMK2 in TOM-1 and BV-173 cells could be detected by RT-qPCR and at the protein level by WB. IC50 after LIMK1/2 inhibitor exposure alone was 580nM in TOM-1 cells and 1000nM in BV-173 cells. All combination experiments with the LIMK1/2 inhibitor and imatinib, dasatinib, nilotinib and ponatinib yielded synergistic CI for treatment of both TOM-1 and BV-173 cell lines. Cell cycle arrest in the G1/S transition was detected and LIMK1/2 inhibition induced dose dependent apoptosis in TOM-1 and BV-173 cells up to 40% at doses <1000nM. Upon treatment with the LIMK1/2 inhibitor, decrease of LIMK1 protein expression could be detected by WB, while LIMK2 expression was left unaffected. In both cell lines, LIMK1/2 inhibitor exposure lead to activating downstream dephosphorylation of cofilin as expected. No significant toxicity of increasing doses of LIMK1/2 inhibitor after exposure of CD34+ cells from healthy donors could be detected. To test the in vivo activity of LIMK1/2 inhibition, C57Bl/6 mice were transplanted with CDKN2Ako/BCR-ABL1+ B-ALL cells. Leukemic mice were treated with LIMK1/2 inhibitor alone, nilotinib or combination of LIMK1/2 inhibitor and nilotinib compared to untreated mice. The combination of nilotinib and LIMK1/2 inhibitor significantly delayed the appearance of leukemic cells in PB as detected by GFP+ cells once weekly or at death if possible with mice considered having leukemia if >1% GFP+ cells were detected in PB. Furthermore, nilotinib+LIMK1/2 inhibitor prolonged significantly the survival of mice compared to either nilotinib (p=0.0006) or LIMK1/2 inhibitor alone and untreated mice (p<0.0001) (Figure 1). Conclusion: Combination of LIMK1/2 inhibitor with BCR-ABL targeting TKI is synergistic and has significant anti-leukemic activity in BCR-ABL1+ ALL in vitro and in vivo models. Disclosures Braun: CELLIPSE: Research Funding. Prudent:CELLIPSE: Employment. Paublant:CELLIPSE: Employment. Baruchel:Jazz Pharmaceuticals: Consultancy, Honoraria, Other: Travel, accommodations or expenses; Shire: Research Funding; Servier: Consultancy; Amgen: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Roche: Consultancy. Dombret:CELLIPSE: Research Funding.

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