The Protein Tyrosine Phosphatase STS-1 Interacts with Bcr-Abl and Impairs the Response of Philadelphia-Positive Acute Lymphoblastic Leukemia (ALL) to Tyrosine Kinase Inhibitors

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3236-3236
Author(s):  
Marcus Liebermann ◽  
Daniela Hoeller ◽  
Susanne Badura ◽  
Tamara Tesanovic ◽  
Hubert Serve ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Research Funding ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Signalling Pathways ◽  
Abl Kinase ◽  
Clinical Resistance

Abstract Abstract 3236 Bcr-Abl is a leukemogenic fusion gene that by itself is sufficient for cellular transformation (Daley et al.) and is the hallmark of chronic myeloid leukemia and Philadelphia chromosome positive (Ph+) ALL. The Bcr-Abl fusion protein is a constitutively active tyrosine kinase (TK) which disrupts multiple cellular signalling pathways controlling apoptosis, cell cycle, proliferation and DNA repair. In Ph+ ALL, a subtype of ALL with a particularly poor prognosis, targeted inhibition of Bcr-Abl activity by Abl kinase inhibitors such as imatinib has improved treatment outcome but has not abrogated the frequent development of clinical resistance. In addition to mutations in the Bcr-Abl tyrosine kinase domain (TKD), it has become apparent that other resistance mechanisms contribute to disease progression. The activity of proteins involved in the above-mentioned signalling pathways and possibly resistance to TK inhibitors (TKI) is controlled at least partially by posttranslational modifications such as phosphorylation, which is regulated by the balance between kinases and protein tyrosine phosphatases (PTP). We previously showed that PTP1B is a negative regulator of Bcr-Abl-mediated transformation and modulates sensitivity to the TKI imatinib (Koyama et al). We hypothesized that other phosphatases for which Bcr-Abl is a substrate may also contribute to resistance, one candidate being Suppressor of T-cell receptor Signalling 1 (STS-1), which negatively regulates the endocytosis of receptor TK involved in a variety of hematologic malignancies. It was the aim of this study to determine whether: i) Bcr-Abl is a substrate of STS-1 ii) STS-1 is able to dephosphorylate Bcr-Abl iii) expression of STS-1 reduces the proliferation of Bcr-Abl expressing cells by inhibiting Bcr-Abl kinase activity iv) the level of STS-1 expression modulates the sensitivity of Bcr-Abl positive cells to TKI In order to answer these questions, we used 293T cells, a human primary embryonal kidney cell line, and the IL3-dependent murine pro B cell line Ba/F3. Both cell lines were modified with constructs encoding both forms of Bcr-Abl (p185/p210) and Sts-1. For experiments with endogenous Bcr-Abl (p185) and Sts-1 we used Sup B15 cells, a human B cell precursor leukemia, and its TKI-resistant subline (Sup B15 RT), which was generated in our lab and is highly resistant not only to imatinib but also to 2nd generation TKIs (Nilotinib & Dasatinib), with no evidence of TKD mutations or transcriptional up-regulation of Bcr-Abl. In all above described cell lines the interaction between Bcr-Abl and Sts-1 could be shown in an overexpressed system (293T & Ba/F3) and on an endogenous level (Sup B15 & Sup B15 RT) by using co-IPs followed by SDS-PAGE and Western blotting. The functional relevance was examined by testing the ability of Sts-1 to dephosphorylate Bcr-Abl. Complete dephosphorylation of Bcr-Abl was shown for p185bcr-abl and p210bcr-abl in 293T cells. To verify that the functional activity was also present in hematopoietic cells, we analyzed the ability of Sts-1 to dephosphorylate Bcr-Abl in Ba/F3 and Sup B15 cells. Dephosphorylation was observed in both cell lines but was less pronounced than in 293T cells. We therefore more closely examined the most important tyrosine (Tyr) residues of Bcr-Abl and identified Tyr245 and Tyr412 as the major targets of Sts-1. Phosphorylation of Tyr245 and Tyr412 was decreased by ∼60% in Ba/F3 cells and ∼39% in Sup B15 cells. These two residues are known to be important for regulating cell proliferation, survival and cell motility. In a competitive proliferation assay in the absence of IL3, the proliferation rate of BA/F3 cells infected with Bcr-Abl and Sts – 1 was reduced compared to a Bcr-Abl infected control population. When treated with imatinib the Sts-1 expressing cells showed an approximately 5-fold reduced proliferation rate compared to cells lacking Sts-1, or to imatinib-resistant cells harbouring the Bcr-Abl “gatekeeper mutation” T315I. The expression level of Sts-1 was found to be approximately 3-fold lower in the Sup B15 RT compared to the WT cell line. Regulation appeared to occur at the transcriptional level as shown by quantitive RT-PCR. These results show that Bcr-Abl is a substrate of Sts-1, that this phosphatase modulates Bcr-Abl kinase activity and may abrogate the response to TKI. This suggests that phosphatases may contribute to the development of clinical resistance of Ph+ leukemias to TKIs. Disclosures: Ottmann: Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding.

scholarly journals Activation of AKT/mTOR Pathway in Ph+ Acute Lymphoblastic Leukemia (ALL) Leads to Non-Mutational Resistance

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2570-2570 ◽  
Author(s):  
Afsar Ali Mian ◽  
Usva Zafar ◽  
Oliver Ottmann ◽  
Martin Ruthardt ◽  
El-Nasir M A Lalani
Keyword(s):  
Tyrosine Kinase ◽  
Cell Line ◽  
Research Funding ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Kinase Domain ◽  
Pi3 Kinase ◽  
Mtor Pathway ◽  
Abl Kinase ◽  
Technology Mutation

Introduction: The t(9;22) (q34;q11) translocation results in the constative active BCR/ABL tyrosine kinase. Der22 involves the Breakpoint Cluster Region (BCR) gene locus with two principal breaks: a. M-bcr, encoding for the p210-BCR/ABL and b. m-bcr, encoding for the p185-BCR/ABL fusion proteins, respectively. BCR/ABL is the oncogenic driver of Chronic Myeloid Leukemia (CML) and 30% of adult Acute Lymphatic Leukemia (ALL). Activated BCR/ABL kinase is responsible for aberrant activation of multiple signaling pathways, such as JAK/STAT, PI3K/AKT and RAS/MAPK which eventually result in leukemic transformation. Successful targeting of BCR/ABL by selective tyrosine kinase inhibitors (TKIs) such as Imatinib, Nilotinib, Dasatinib and Ponatinib are used for the treatment of Philadelphia chromosome-positive (Ph+) leukemias. Most patients with CML in the early stage (CML-CP) treated with TKIs have increased overall survival. However, TKIs have not been as effective in patients with CML blast crisis (CML-BC) or Ph+ ALL. Point mutations in the tyrosine kinase domain (TKD) of BCR/ABL have emerged as the predominant cause of acquired resistance. These mutations are observed in up to 80% of patients with CML-BC and Ph+ ALL and in ~ 50% of Imatinib-resistant patients. In the remaining 20-50% of patients the mechanism of resistance to TKIs remains elusive. The aim of this study was to investigate the mechanism of non-mutational resistance in Ph+ ALL. Methods: As models for non-mutational resistance, we used patient derived long term cultures (PDLTCs) from Ph+ ALL patients with different levels of non-mutational drug resistance and the SupB15RT, a Ph+ ALL cell-line rendered resistant by exposure to increasing doses of Imatinib and cross-resistant against all approved ABL Kinase Inhibitors (AKIs). Cell proliferation was assessed by XTT/MTT and trypan blue dye exclusion. Signaling pathway proteins were assessed by Western Blot analysis. Chromosomal karyotyping was undertaken on single cell genomes using multi-color FISH (M-FISH) technology. Mutation analysis on the ABL kinase domain was done by sequencing the heminested PCR products obtained from SupB15-WT and SupB15RT cell-lines. Results: A non-mutational resistance cell line SupB15RT, was developed by exposing SupB15 cells to an increasing concentration of Imatinib over a 3 month period. SupB15RT were able to grow in 10 µM Imatinib. SupB15RT cells were karyotypically and mutationaly identical to SupB15 WT. All approved AKIs and allosteric inhibitors like GNF-2, ABL001 and Crizotinib were unable to inhibit growth of these cells, except for Dasatinib (IC50 40nM), a multi-target kinase inhibitor. Experiments to determine the mode of resistance revealed high level (3 fold) of activation of AKT/mTOR enabling these cells to grow and proliferate. We targeted the AKT/mTOR pathway using BKM-120 (PI3 Kinase inhibitor), BEZ-235 (PI3 Kinase and mTOR pathway) and Trorin1/Torin2 (mTORC1 and mTORC2) and found that Torin-1 and Torin-2 significantly inhibited proliferation of SupB15RT, in a dose dependent manner, with an IC50 of 11-20 nM. As Dasatinib alone inhibited growth of SupB15RT cells at 40-50nm concentrations, we combined Dasatinib with Torin1 and found that the combination of these two compounds had an additive inhibitory effect on cell growth. Following this we examined clinical samples from patients. We used three different Ph+ PDLTCs: a. HP (BCR/ABL negative), b. PH (BCR/ABL positive and responsive to TKIs) and c. BV (BCR/ABL positive and non-mutational resistant to TKIs). Interestingly, we found that AKT/mTOR pathway was activated in BV cells and its proliferation was inhibited by Torin1 with IC-50 of 50nM. Conclusion: Our experiments revealed an additional pathway involved in the evolution of non-mutational resistance in Ph+ ALL which could assist in developing novel targeted therapy for Ph+ ALL patient(s) with non-mutational resistance. Disclosures Ottmann: Celgene: Honoraria, Research Funding; Incyte: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Novartis: Honoraria; Takeda: Honoraria; Fusion Pharma: Honoraria; Pfizer: Honoraria; Roche: Honoraria.

Blocking of Cytokine Survival Signals along with Intense Bcr-Abl Kinase Inhibition May Eradicate CML Progenitor Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3250-3250
Author(s):  
Devendra K Hiwase ◽  
Deborah L White ◽  
Jason A Powell ◽  
Verity A Saunders ◽  
Stephanie Zrim ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Research Funding ◽  
Kinase Activity ◽  
Short Term ◽  
Kinase Inhibition ◽  
Gm Csf ◽  
Abl Kinase ◽  
Cd34 Cells ◽  
Short Term Culture

Abstract Abstract 3250 Poster Board III-1 Preclinical studies of imatinib set the paradigm of continuous Bcr-Abl kinase inhibition for optimal response in chronic myeloid leukemia (CML). However, the clinical success of once daily dasatinib, despite its short serum half life, implies that intermittent inhibition of Bcr-Abl kinase activity is sufficient for clinical response. In vitro studies also demonstrated that short-term intense (≥90%) Bcr-Abl kinase inhibition triggers cell death in BCR-ABL + cell lines, demonstrating their oncogene addiction. However, the effect of short-term intense kinase inhibition on CD34+ CML progenitors is not studied. Clinical, mathematical modelling and in vitro studies suggest that leukemic stem cells (LSC) are difficult to eradicate and hence the majority of CML patients may not be cured with tyrosine kinase inhibitors (TKI). Inadequate Bcr-Abl kinase inhibition has been postulated to cause refractoriness of LSC to TKI's. This may be due to increased expression of ABCB1 and ABCG2 efflux proteins, or the quiescent state of LSC. However, the phenomenon could be independent of Bcr-Abl kinase activity. In vivo leukemic progenitors live in a cytokine rich environment which may be providing a mechanism for Bcr-Abl independent resistance. We have assessed the impact of short-term intense Bcr-Abl kinase inhibition on CML cell lines and CML CD34+ primary cells in the presence and absence of cytokines. In CML cell lines, short-term (cells were cultured with dasatinib for 30 min and following thorough drug washout, cells were recultured in drug free media for 72 hr) intense Bcr-Abl kinase inhibition with 100 nM dasatinib triggers cell death. In CML-CD34+ cells 30 min of culture with 100 nM dasatinib (n=13) or 30 μM IM (n=7) reduced the level of p-Crkl (surrogate marker of Bcr-Abl kinase activity) by 97±3% and 96±4% respectively. In the presence of either a six growth factors cocktail (6-GF; n=10) or GM-CSF (n=11) or G-CSF (n=4) alone, despite 97% inhibition of p-Crkl, short-term culture with 100 nM dasatinib (D100ST) reduced colony forming cells (CFC) by only 24%, 32% or 5%, respectively. However without cytokines, D100ST reduced CML-CD34+ CFCs by 70%. Consistent with the results observed with dasatinib, short-term culture with 30 μM imatinib (IM) (n=3) also reduced 90% CFC in the absence of cytokines but by only 38% in the presence of 6-GF. These results suggest that in CML-CD34+ cells, GM-CSF, G-CSF or 6-GF mediate Bcr-Abl independent TKI resistance. It is possible that cytokines may be promoting cell survival via signalling pathways that are refractory to dasatinib. To examine this possibility, we assessed the effect of D100ST on p-STAT5 signalling in CML-CD34+ cells, in the presence and absence of GM-CSF, G-CSF or 6-GF. STAT5 was constitutively phosphorylated in CML-CD34+ cells, and in the absence of cytokines, D100ST reduced the p-STAT 5. STAT5 phosphorylation was not inhibited by D100ST when cells were cultured with 6-GFs or GM-CSF however, the combination of D100ST and a Janus kinase (Jak) inhibitor dramatically reduced p-STAT5. Similarly, in the presence of GM-CSF (32.35±5.16% vs. 68.33±14.90%) or G-CSF (58.13±13 vs. 94.68±21.12) combination of D100ST and JAK inhibitor significantly reduced CFC compared to D100ST only. Thus our data suggest that in contrast to CML cell lines, primary CML progenitors may not be completely dependent on the BCR-ABL oncogene and that activation of the cytokine mediated JAK-2/STAT-5 pathway may circumvent the need for BCR-ABL signalling for maintenance of survival. Thus a therapeutic strategy based on short-term intense kinase inhibition may have limited success unless critical redundant cytokine-induced survival pathways are also inhibited. We postulate that blockade of cytokine signalling along with short-term intense Bcr-Abl kinase inhibition with a potent second generation TKI may provide a novel strategy to eradicate primitive CML cells. Fig 1 In CML-CD34+ cells, Jak kinase inhibition abrogates the rescuing effect of cytokines on cell death induced by BCR-ABL blockade: In the absence of cytokines (No GF, n=11) short-term culture with 100 nM dasatinib (D100ST) reduced CFCs by 67% of control, however in the presence of 6-GFs (n=10), GM-CSF (n=10) or G-CSF (n=4) it could reduce CFCs by only 24%, 32% or 5% of control respectively (B) In the presence of GM-CSF (n= 4) or G-CSF (n= 4), combination of Jak inhibition and D100ST reduced CFC compared to dasatinib alone. Fig 1. In CML-CD34+ cells, Jak kinase inhibition abrogates the rescuing effect of cytokines on cell death induced by BCR-ABL blockade: In the absence of cytokines (No GF, n=11) short-term culture with 100 nM dasatinib (D100ST) reduced CFCs by 67% of control, however in the presence of 6-GFs (n=10), GM-CSF (n=10) or G-CSF (n=4) it could reduce CFCs by only 24%, 32% or 5% of control respectively (B) In the presence of GM-CSF (n= 4) or G-CSF (n= 4), combination of Jak inhibition and D100ST reduced CFC compared to dasatinib alone. Disclosures: White: Novartis and Britol-Myers Squibb: Research Funding. Hughes:BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Statin Treatment Prevents FLT3 Glycosylation and Overcomes Resistance to FLT3 Tyrosine Kinase Inhibitors

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1421-1421
Author(s):  
Allen Williams ◽  
Li Li ◽  
Bao Nguyen ◽  
Patrick Brown ◽  
Mark J. Levis ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Map Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Cell Lines ◽  
Kinase Inhibitors ◽  
Drug Binding ◽  
Receptor Expression ◽  
Hematopoietic Stem ◽  
Clinical Resistance ◽  
Surface Localization

Abstract Abstract 1421 FLT3 is a receptor tyrosine kinase that is expressed on hematopoietic stem and progenitor cells where it functions in cell differentiation, survival and proliferation. It is also one of the most frequently mutated genes in acute myeloid leukemia (AML), and has thus become a target for modulation by the use of FLT3 tyrosine kinase inhibitors (TKI). Unfortunately, clinical resistance emanating via several different mechanisms has limited the potential benefit of FLT3 TKI. Some of these include FLT3 mutations that reduce drug binding, elevated FLT3 ligand (FL) levels that shift the dose-response curve and activation of parallel signaling pathways. These all pose major challenges to TKI effectiveness that must be overcome to improve patient outcomes. Since FLT3 is dependent upon N-linked glycosylation for its maturation and surface localization, we sought to determine whether statins might disrupt FLT3 signaling. Statins inhibit the mevalonate pathway and reduce levels of all ensuing end-products including dolichol, which transfers preassembled oligosaccharides onto nascent polypeptides. Here, we demonstrate by Western blotting that statins can indeed prevent complex FLT3 glycosylation, thus leading to loss of surface receptor expression. Immunofluorescence microscopy confirms a reduction in surface localization and a concomitant increase in intracellular FLT3/ITD accumulation. Interestingly, this aberrant localization was associated with increased STAT5 activation while inhibiting both MAP kinase and AKT phosphorylation. We have extended our previous findings that statins are cytotoxic to mutant FLT3 expressing cell lines to examine whether they are also able to overcome the resistance mechanisms discussed above. We show that the following mechanisms of resistance could be circumvented by fluvastatin. First, stimulation of BaF3 FLT3/ITD cells with exogenous FL at physiologic concentrations induces a three-fold increase in the IC50 for inhibition of phosphorylated FLT3/ITD by the FLT3 TKI lestaurtinib. Pretreatment with fluvastatin for 24 h eliminated surface FLT3/ITD: FL interactions and restored the potency of lestaurtinib. Secondly, resistance to another clinical FLT3 TKI, sorafenib, caused by the FLT3/ITD N676K or D835Y mutations increased the IC50 from 5 nM for BaF3/ITD cells to 30 nM and >50 nM, respectively. In contrast, both of these mutants remained just as sensitive to treatment with fluvastatin as were the BaF3/ITD cells (approx. IC50 of 0.2 μM). A third mechanism of resistance that can be circumvented by fluvastatin is the over expression and/or activation of alternative pathways that can compensate for inhibited FLT3. A model of this is rescue of BaF3 FLT3/ITD cell lines from FLT3 TKI-mediated cytotoxicity by stimulation with IL-3. When this cell line is stimulated with IL-3, fluvastatin is able to inhibit activation of STAT5, AKT and MAP kinase caused by the IL-3 receptor, as well as FLT3/ITD signaling, leading to cell death. Fluvastatin was also effective in other cell lines expressing constitutively activated FLT3 (Molm-14, MV4;11, HB1119 and SEM-K2 cells) and in other cell lines in which glycosylated transmembrane receptors drive proliferation (mutant c-Kit in Kasumi cells) but not in cells that rely upon intracellular kinases (BCR-ABL in K562 cells). Importantly, fluvastatin also reduced FLT3 glycosylation in and was cytotoxic to primary AML patient samples harboring FLT3/ITD mutations at therapeutically achievable concentrations (1 μM). Finally, fluvastatin reduced engraftment of BaF3 FLT3/ITD cells transplanted in syngeneic Balb/c mice and prolonged their survival. These results demonstrate that statins, a class of drugs already FDA approved, might be useful, either alone or in combination with a FLT3 TKI, in the management of FLT3 AML cases including those resistant to FLT3 TKI. Disclosures: Levis: Ambit Biosciences, Inc: Consultancy.

Treating Ph+ Acute Lymphoblastic Leukemia (ALL) in the Elderly: The Sequence of Two Tyrosine Kinase Inhibitors (TKI) (Nilotinib and Imatinib) Does Not Prevent Mutations and Relapse.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2601-2601 ◽  
Author(s):  
Cristina Papayannidis ◽  
Paola Fazi ◽  
Alfonso Piciocchi ◽  
Francesco Di Raimondo ◽  
Giovanni Pizzolo ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Tyrosine Kinase ◽  
Research Funding ◽  
Kinase Inhibitors ◽  
Lymphoblastic Leukemia ◽  
The Elderly ◽  
Kinase Domain ◽  
Intensive Chemotherapy ◽  
Molecular Responses ◽  
Abl Kinase

Abstract Abstract 2601 Background: Tyrosine Kinase Inhibitors (TKI) have been shown to be very effective for the treatment of Acute Lymphoblastic Leukemia (ALL), with a Complete Hematologic Remission (CHR) rate close to 100%, and a high rate of Complete Cytogenetic and Molecular responses (CCgR and CMR). However, when they are used alone, as single agents, most patients relapse, so that they are currently used in combination with chemotherapy and as a preparation to allogeneic stem cell transplantation (SCT). Since Ph+ ALL is more frequent in the elderly, many patients cannot tolerate intensive chemotherapy and are not eligible for SCT. We have explored if the administration of two TKIs, Nilotinib (NIL) and Imatinib (IM) can improve the results without increasing the toxicity. Aims: To evaluate the response and the outcome of Ph+ ALL patients treated with the sequential administration of NIL and IM, to investigate the type and number of BCR-ABL kinase domain mutations developing during and after the study. Methods: We have designed a study (ClinicalTrials.gov. NCT01025505) in which patients more than 60 years old or unfit for intensive chemotherapy and SCT where treated with two TKIs, NIL 400 mg twice daily, and IM 300 mg twice daily, alternating for 6 weeks for a minimum of 24 weeks (study core) and indefinitely in case of response. The 6-weeks rotation schedule was respected, irrespectively of temporary discontinuations. The primary end-point was the rate of Disease Free Survival (DFS) at 24 weeks (4 courses of treatment); the secondary end points included the evaluation of CHR, CCgR and CMR rates. Mutation analysis was performed by nested RT-PCR amplification of the ABL kinase domain of the BCR-ABL transcript (codons 206 through 421). Amplified products were screened by denaturing-high performance liquid chromatography (D-HPLC). Samples scored positive for the presence of sequence variations were then subjected to direct automatic sequencing to characterize the mutation. Results: 39 patients have been enrolled in 15 Italian hematologic Centers (median age 66 years, range 28–84). Among these, 8 patients were unfit for standard chemotherapy or SCT (median age 50 years, range 28–59). 27 patients were p190, 5 were p210 and 7 were p190/p210. After 6 weeks of treatment, 36 patients were evaluable for response: 34 were in CHR (94%) and 2 in PHR (6%). 23 patients have already completed the study core (24 weeks), 87% were in CHR and 17 are currently continuing therapy in the protocol extension phase. Thus, the OS at 1 year is 79%, and 64% at 2 years. Overall, 1 patient was primarily resistant and 13 patients have relapsed, with a median time to relapse of 7.6 months (range 0.8–16.1 months), for a DFS of 51.3% at 12 months (Figure 1). Mutations detected were T315I in 2 cases, Y253H in 3 cases, T315I and Y253H in 1 case, E255K in 1 case, T315I and E255K in 1 case, E255V and Y253H in 1 case. Two patients were WT. A detailed kinetics of Molecular responses is shown in Table 1. Data on mutational analysis are reported in Table 2. Further details about Cytogenetic and Molecular responses, and about Adverse Events will be provided on site. Conclusions: In this small cohort of Ph+ ALL elderly/unfit patients, the rates of relapse and progression were not likely to be different from the rates observed with Imatinib alone (Vignetti et al, Blood 2007, May 1;109(9):3676-8) and Dasatinib alone (Foà, Blood 2011, Dec 15;118(25):6521-8). It's important to notice that the mutations that occurred at the time of relapse were sensitive to other TKIs (Dasatinib and Ponatinib). Acknowledgments: COFIN, Bologna University, BolognAIL, PRIN, Fondazione del Monte di Bologna e Ravenna, INPDAP. Disclosures: Pizzolo: Hoffmann-La Roche: Consultancy, Honoraria. Luppi:CELGENE CORPORATION: Research Funding. Vallisa:CELGENE CORPORATION: Research Funding. Martinelli:NOVARTIS: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy. Baccarani:ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau.

Autophagic and Apoptotic Effects of Tyrosine Kinase Inhibitors in Myeloid Leukemia: Comparison of Three Generation

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4915-4915
Author(s):  
Cagla Kayabasi ◽  
Cigir Biray Avci ◽  
Sunde Yilmaz Susluer ◽  
Tugce Balci ◽  
Yusuf Baran ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Tyrosine Kinase ◽  
Cell Line ◽  
Tyrosine Kinase Inhibitors ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Fluorescent Protein ◽  
Control Group ◽  
Green Fluorescent

Abstract Abstract 4915 The aim of the study was to evaluate the differences in cytotoxicity, apoptosis and autophagy levels in myeloid leukemia cell lines treated with tyrosine kinase inhibitors compared to cell line resistant to imatinib and control group. Chronic myeloid leukemia model was created by using cell lines as K-562 cell line for Ph+ chronic myeloid leukemia model, HL-60 cell line for acute promyelocytic Ph- leukemia model. NCI-BL2171 normal cell line was used as a control group while K562/ima3 cell line was used as an imatinib resistant model. Imatinib (STI571), Dasatinib (BMS-354825), Ponatinib (AP24534) were used as tyrosine kinase inhibitors in this study. Cytotoxicity analysis was conducted by WST-1 analysis. Apoptotis was evaluated by AnnexinV-enhanced green fluorescent protein (EGFP) and by Mitoprobe JC-1 for Mitochondrial Potential Detection. Autophagy was analyzed by The Premo Autophagy Tb/GFP TR-FRET LC3B assay which measures autophagy in cells expressing green fluorescent protein (GFP)-tagged LC3B using a Tb-based TR-FRET immunoassay approach. By using IC50 doses of tyrosine kinase inhibitors, autophagic effect of these drugs on cell lines were examined at 24th hours. Cells not treated with the active substance or chloroquine were considered as control groups. Chloroquine-treated cells were used as positive control for autophagy. LC3B-II increase is an indicator of autophagic suppression. Cells treated with chloroquine were compared with cells treated with active substances and concentrations of BacMam that displayed the highest LC3B-II increase were selected. Autophagic suppression ratio of the drugs was evaluated among the control group. Cytotoxicity, apoptosis and autophagy analysis results were provided in Table. Compared to control group, 30 μM chloroquine repressed autophagy 1. 93, 1. 48, 2. 74 and 1. 54 fold in K562, HL-60, K562/ima3 and NCI-BL 2171 cells, respectively. In HL-60 cells while Imatinib represented 0. 77 fold autophagy, it repressed autophagy 1. 77 and 3. 49 fold in K562 and K562/ima3 cells respectively. Dasatinib repressed autophagy 2. 11, 1. 95 and 4. 62 fold and Ponatinib repressed autophagy 2. 09, 1. 60 and 9. 15 fold in K562, HL-60, K562/ima3 cells respectively. Imatinib, Dasatinib and Ponatinib did not repressed autophagy in NCI-BL 2171 cells. In conclusion, apoptosis and autophagy paradox was illuminated in myeloid leukemia cells via tyrosine kinase inhibitors and autophagy may be a new strategy for targeted therapy in myeloid leukemia after clarifying responsible genes and proteins in signal transduction pathways. Cytotoxicity Apoptosis Autophagy WST-1 IC50 (nM) Annexin V JC-1 Premo Autophagy Ýmatinib Dasatinib Ponatinib Ýmatinib Dasatinib Ponatinib Ýmatinib Dasatinib Ponatinib Ýmatinib Dasatinib Ponatinib K562 24th hour 1.70 3.65 3.05 3.07 1.37 1.35 1.43 1.77 2.11 2.09 48th hour 650.00 0.24 2.67 2.51 2.32 2.03 2.35 2.06 72nd hour 4.53 4.81 3.00 2.97 3.07 2.50 HL-60 24th hour 1.33 1.26 1.32 1.29 1.22 1.34 0.77 1.95 1.60 48th hour 18000.00 1.39 1.23 1.41 1.61 1.92 1.96 72nd hour 896.00 607.00 2.21 1.80 2.82 1.58 1.73 2.23 K562/ima3 24th hour 1.33 0.76 1.69 1.51 1.36 1.59 3.49 4.62 9.15 48th hour 18350.00 1830.00 9.87 1.80 1.94 2.03 2.82 1.22 1.40 72nd hour 1.34 1.44 1.41 2.61 1.40 1.56 NCI-BL 2171 24th hour 48.00 2.48 2.79 2.62 3.99 4.04 4.25 1.01 0.88 0.90 48th hour 274.00 30.00 4.11 4.33 4.15 5.05 2.75 3.11 72nd hour 6.14 6.04 6.03 8.27 3.71 3.95 Disclosures: No relevant conflicts of interest to declare.

scholarly journals MYD88 L265P Augments Proximal B-Cell Receptor Signaling in Large B-Cell Lymphomas Via an Interaction with DOCK8

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1324-1324
Author(s):  
Elisa Mandato ◽  
Qingsheng Yan ◽  
Jing Ouyang ◽  
Julia Paczkowska ◽  
Yan Qin ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
B Cell ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Receptor ◽  
Genetically Engineered ◽  
Bcr Signaling ◽  
Myd88 L265p ◽  
Research Grant

Abstract Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous disease comprised of five subtypes including a subset of poor-prognosis activated B cell (ABC)-enriched tumors with frequent MYD88L265P mutations, often in association with CD79B alterations (Cluster 5 DLBCLs) (Nat. Med. 2018; 24:679-690). Primary central nervous system lymphomas (PCNSLs) and primary testicular lymphomas (PTLs) have similar genetic signatures including recurrent MYD88L265P mutations and concurrent CD79B alterations (Blood 2016; 127: 869-81). These findings prompted us to evaluate a potential role for MYD88L265P in proximal B-cell receptor (BCR) signaling, in addition to its defined function as an intermediary in the Toll-Like Receptor (TLR) pathway and downstream NF-kB activation. In previous studies by Jabara et al., wild-type (WT) MYD88 was found to be constitutively associated with the DOCK8 adapter and the PYK2 tyrosine kinase in normal B-cells (Nat. Immunol. 2012; 13:612-20). In this setting, physiologic ligation of TLR9 with CpG oligodeoxynucleotides (CpG) induced PYK2-mediated phosphorylation of DOCK8, recruitment of Src kinases, including LYN, and downstream activation of the proximal BCR pathway member, spleen tyrosine kinase (SYK) (Nat. Immunol. 2012; 13:612-20). We postulated that mutated MYD88L265P might similarly augment proximal BCR signaling in DLBCLs in the absence of physiologic (CpG-induced) TLR9 signaling. Using three DLBCL cell lines (OCI-Ly1, SU-DHL4 and OCI-Ly7) with intact BCR signaling and WT endogenous MYD88 and CD79B, we first established that physiologic CpG activation of TLR signaling induced the phosphorylation of PYK2 and the proximal BCR signaling components, SYK and Bruton's tyrosine kinase (BTK). Thereafter, we genetically engineered these three DLBCL cell lines to express MYD88 L265P or MYD88 WT, alone or in association with CD79B Y196F. In all three cell lines, the co-expression of MYD88 L265P and CD79B Y196F significantly increased magnitude and duration of SYK and BTK phosphorylation following BCR crosslinking. These findings highlight the likely role of MYD88L265P in CD79BY196F-associated proximal BCR signaling in DLBCL. To elucidate the potential role of the DOCK8 adapter in MYD88 L265P-augmented BCR signaling, we first assessed the colocalization of MYD88 WT or MYD88 L265P with DOCK8 in the same three genetically engineered DLBCL cell lines using proximity ligation assays (PLA), which detect protein-protein interactions at less than 40 nm in situ. In each of these cell lines, we detected significantly increased co-localized MYD88 L265P/DOCK8 signals in comparison to MYD88 WT/DOCK8 signals (p<.0001, all). Additionally, there were significantly increased co-localized DOCK8/LYN signals in DLBCL cell lines that expressed MYD88 L265P rather than MYD88 WT (p<.0001, all). These data provide the first direct evidence of an enhanced association between MYD88 L265P, DOCK8 and LYN in BCR-dependent DLBCLs and a basis for enhanced BCR signaling in primary tumors with concurrent MYD88L265P and CD79B genetic alterations. We next analyzed the consequences of MYD88 L265P-associated, DOCK8-dependent increased proximal BCR signaling by depleting DOCK8 in BCR-dependent DLBCL cells with endogenous MYD88L265P/CD79BY196F alterations (HBL1 and TMD8) or endogenous unmutated MYD88 WT/CD79B WT (OCI-Ly1 and SU-DHL4). ShRNA-mediated DOCK8 knockdown (KD) significantly decreased BCR-mediated phosphorylation of SYK and BTK in MYD88L265P/CD79BY196F DLBCL cell lines but not in lines with MYD88 WT/CD79B WT, highlighting the specific role of DOCK8 in MYD88 L265P-associated proximal BCR signaling. Of great interest, DOCK8 KD selectively decreased the proliferation of MYD88L265P/CD79BY196F, but not MYD88WT/CD79BWT, DLBCLs (p<.004, HBL1 and p<.009, TMD8; p = non sig., OCI-Ly1 and SU-DHL4). Additionally, DOCK8 KD significantly increased the efficacy of chemical PI3Kα/δ (copanlisib) and BTK (ibrutinib) inhibition in MYD88L265P/CD79BY196F DLBCLs (HBL1 and TMD8). Taken together, these data identify DOCK8 as an intermediary in MYD88L265P-driven proximal BCR signaling and a possible treatment target in LBCLs with co-occurring MYD88L265P/CD79BY196F mutations. Disclosures Shipp: AstraZeneca: Consultancy, Research Funding; Immunitas Therapeutics: Consultancy; Bristol Myers Squibb: Research Funding; Merck: Research Funding; Bayer: Other: Institution: Research Grant/Funding; Abbvie: Other: Institution: Research Grant/Funding.

scholarly journals Targeted Inhibition of PI3K α/δ Is Synergistic with BCL-2 Blockade in Genetically Defined Subtypes of DLBCL

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 39-39
Author(s):  
Kamil Bojarczuk ◽  
Kirsty Wienand ◽  
Jeremy A. Ryan ◽  
Linfeng Chen ◽  
Mariana Villalobos-Ortiz ◽  
...  
Keyword(s):  
Tumor Growth ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Research Funding ◽  
Single Agent ◽  
Research Report ◽  
Genetic Bases

Abstract Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous disease that is transcriptionally classified into germinal center B-cell (GCB) and activated B-cell (ABC) subtypes. A subset of both GCB- and ABC-DLBCLs are dependent on B-cell receptor (BCR) signaling. Previously, we defined distinct BCR/PI3K-mediated survival pathways and subtype-specific apoptotic mechanisms in BCR-dependent DLBCLs (Cancer Cell 2013 23:826). In BCR-dependent DLBCLs with low baseline NF-κB activity (GCB tumors), targeted inhibition or genetic depletion of BCR/PI3K pathway components induced expression of the pro-apoptotic HRK protein. In BCR-dependent DLBCLs with high NF-κB activity (ABC tumors), BCR/PI3K inhibition decreased expression of the anti-apoptotic NF-κB target gene, BFL1. Our recent analyses revealed genetic bases for perturbed BCR/PI3K signaling and defined poor prognosis DLBCL subsets with discrete BCR/PI3K/TLR pathway alterations (Nat Med 2018 24:679). Cluster 3 DLBCLs (largely GCB tumors) exhibited frequent PTEN deletions/mutations and GNA13 mutations. Cluster 5 DLBCLs (largely ABC tumors) had frequent MYD88L265P and CD79B mutations that often occurred together. These DLBCL subtypes also had different genetic mechanisms for deregulated BCL2 expression - BCL2 translocations in Cluster 3 and focal (18q21.33) or arm level (18q) BCL2 copy number gains in Cluster 5. These observations prompted us to explore the activity of PI3K inhibitors and BCL2 blockade in genetically defined DLBCLs. We utilized a panel of 10 well characterized DLBCL cell line models, a subset of which exhibited hallmark genetic features of Cluster 3 and Cluster 5. We first evaluated the cytotoxic activity of isoform-specific, dual PI3Kα/δ and pan-PI3K inhibitors. In in vitro assays, the PI3Kα/δ inhibitor, copanlisib, exhibited the highest cytotoxicity in all BCR-dependent DLBCLs. We next assessed the transcriptional abundance of BCL2 family genes in the DLBCLs following copanlisib treatment. In BCR-dependent GCB-DLBCLs, there was highly significant induction of the pro-apoptotic HRK. In BCR-dependent ABC-DLBCLs, we observed significant down-regulation of the anti-apoptotic BFL1 protein and another NF-κB target gene, BCLxL (the anti-apoptotic partner of HRK). We then used BH3 profiling, to identify dependencies on certain BCL2 family members and to correlate these data with sensitivity to copanlisib. BCLxL dependency significantly correlated with sensitivity to copanlisib. Importantly, the BCLxL dependency was highest in DLBCL cell lines that exhibited either transcriptional up-regulation of HRK or down-regulation of BCLxL following copanlisib treatment. In all our DLBCL cell lines, PI3Kα/δ inhibition did not alter BCL2 expression. Given the genetic bases for BCL-2 deregulation in a subset of these DLBCLs, we next assessed the activity of the single-agent BCL2 inhibitor, venetoclax, in in vitro cytotoxicity assays. A subset of DLBCL cell lines was partially or completely resistant to venetoclax despite having genetic alterations of BCL2. We postulated that BCR-dependent DLBCLs with structural alterations of BCL2 might exhibit increased sensitivity to combined inhibition of PI3Kα/δ and BCL2 and assessed the cytotoxic activity of copanlisib (0-250 nM) and venetoclax (0-250 nM) in the DLBCL cell line panel. The copanlisib/venetoclax combination was highly synergistic (Chou-Talalay CI<1) in BCR-dependent DLBCL cell lines with genetic bases of BCL2 deregulation. We next assessed copanlisib and venetoclax activity in an in vivo xenograft model using a DLBCL cell line with PTENdel and BCL2 translocation (LY1). In this model, single-agent copanlisib did not delay tumor growth or improve survival. Single-agent venetoclax delayed tumor growth and improved median survival (27 vs 51 days, p<0.0001). Most notably, we found that the combination of copanlisib and venetoclax delayed tumor growth significantly longer than single-agent venetoclax (p<0.0001). Additionally, the combined therapy significantly increased survival in comparison with venetoclax alone (median survival 51 days vs not reached, p<0.0013). Taken together, these results provide in vitro and in vivo pre-clinical evidence for the rational combination of PI3Kα/δ and BCL2 blockade and set the stage for clinical evaluation of copanlisib/venetoclax therapy in patients with genetically defined relapsed/refractory DLBCL. Disclosures Letai: AbbVie: Consultancy, Other: Lab research report; Flash Therapeutics: Equity Ownership; Novartis: Consultancy, Other: Lab research report; Vivid Biosciences: Equity Ownership; AstraZeneca: Consultancy, Other: Lab research report. Shipp:AstraZeneca: Honoraria; Merck: Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Research Funding.

scholarly journals Arsenic Trioxide Synergizes with FLT3 Tyrosine Kinase Inhibitors to Kill FLT3-ITD+ Leukemic Cells through Depressed Production and Degradation of FLT3 Protein

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1568-1568
Author(s):  
Kozo Nagai ◽  
Lihong Hou ◽  
Li Li ◽  
Bao Nguyen ◽  
Courtney M Shirley ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Arsenic Trioxide ◽  
Tyrosine Kinase Inhibitors ◽  
Cell Lines ◽  
Research Funding ◽  
Kinase Inhibitors ◽  
Leukemia Cell ◽  
Leukemic Cells ◽  
Potential Candidate ◽  
Leukemia Cell Lines

Abstract A number of selective FLT3 tyrosine kinase inhibitors (TKIs) have been tested for treatment of FLT3-ITD+ AML. However, monotherapy with FLT3 TKIs alone has achieved only transient and limited clinical responses due to several resistance mechanisms. Arsenic trioxide (ATO) has demonstrated significant efficacy in treating acute promyelocytic leukemia (APL). ATO has also shown some activity in treating non-APL myeloid leukemias. Recent studies have demonstrated that ATO can affect the degradation of oncogenic mutant proteins including mutant p53 and NPM through the ubiquitin-proteasome pathway (UPP). Here we investigated the feasibility of combining FLT3 TKIs with ATO in the treatment of FLT3-ITD+leukemia. Anti-leukemic effects against FLT3-ITD+ patient AML samples and leukemia cell lines (Molm14, MV4;11) by FLT3 TKIs (Sorafenib, quizartinib), ATO and the combination were examined by MTT, apoptosis, cell viability and colony forming assays. Our data revealed that the combination showed synergistic growth inhibition of the FLT3-ITD+ cell lines Molm14 and MV4;11, with combinatorial index (CI) values at ED50 below 1.0 for both cell lines (CI values were 0.46 and 0.56 for ATO + sorafenib, 0.65 and 0.57 for ATO + quizartinib in Molm14 and MV4;11 cells, respectively). In contrast, there was no synergy observed for the combination in treating leukemia cell lines that do not express mutant FLT3. Synergistic effects for the combination in inducing apoptosis and inhibiting colony formation were also observed for the FLT3-ITD+ cell lines. Furthermore, when the combination was used to treat primary FLT3-ITD+ patient samples, there was also significant reduction of viability and clonogenicity. In contrast, normal BM MNCs showed very limited responses to the combination. Western blot (WB) analysis of Molm14 and FLT3-ITD+ patient samples revealed the combination of ATO and sorefenib potently reduced phosphorylation of FLT3 and its downstream targets (STAT5, MAPK, and AKT). In vivoexperiments using the combination to treat NSG mice engrafted with Molm14 cells demonstrated a significant reduction in the level of leukemic cells. We further investigated the mechanism by which ATO contributes to an anti-leukemic effect on FLT3-ITD+ cells. Morphologic and flow cytometric analysis showed that ATO promoted the differentiation of Molm14 cells. The expression of C/EBPα and PU.1, two key regulators for myeloid differentiation, was increased in ATO-treated Molm14 cells at both the mRNA and protein levels. These data suggest ATO is capable of inducing the differentiation of leukemic cells. We also found that, in FLT3-ITD+ cells, ATO decreased expression of FLT3 protein. This could result from reduced FLT3 production and/or increased protein degradation. Further quantitative PCR analysis revealed ATO decreased expression of FLT3 and its upstream regulators HoxA9 and meis1. Co-immunoprecipitation assay showed that ATO facilitated poly-Ubiquitination and degradation of FLT3 in a dose- and time-dependent fashion. These results indicate that ATO exerts its anti-leukemic effects in FLT3-ITD+AML cell lines and primary samples at least partly through reducing the level of FLT3 protein. These studies together demonstrate that ATO has a unique activity towards FLT3-ITD+ leukemia cells. Based on these findings, ATO is a potential candidate to work in combination with FLT3 TKIs to improve the outcome of FLT3-ITD+ AML patients. Disclosures Levis: Millennium: Consultancy, Research Funding; Daiichi-Sankyo: Consultancy, Honoraria; Astellas: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

Survivin silencing sensitizes imatinib-resistant CML cells to the cytotoxic effect of hydroxyurea

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 13119-13119
Author(s):  
F. Stagno ◽  
E. Conte ◽  
S. Stella ◽  
E. Tirrò ◽  
L. Manzella ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Cell Lines ◽  
Cytotoxic Effect ◽  
Kinase Activity ◽  
Point Mutations ◽  
Survivin Expression ◽  
Over Expression ◽  
Abl Kinase ◽  
Abl Tyrosine Kinase ◽  
Apoptosis Protein

13119 Background: Imatinib Mesylate (IM), a semi-specific inhibitor of the BCR-ABL tyrosine kinase, is currently the treatment of choice for Chronic Myeloid Leukemia (CML). However, about one third of CML patients treated with IM develop resistance to the drug because of reactivation of BCR-ABL kinase activity. This phenomenon is usually ascribed to the amplification of the BCR-ABL gene or to the selection of leukemic clones harboring point mutations that abrogate IM binding. To identify novel anti-apoptotic signaling pathways employed by BCR-ABL and devise strategies capable of killing IM-resistant CML cells, we investigated the interplay between BCR-ABL and the Inhibitor of Apoptosis Protein Survivin. Methods: Murine hematopoietic cells (32D) transduced with p210 BCR-ABL and human cell lines either positive (K562, KCL22, KYO1 and LAMA84) or negative (HL60) for the BCR-ABL oncoprotein, were analyzed for Survivin expression by western blot before and after IM treatment. Three different pathways (MAPK, PI3K and JAK2/STA3) potentially involved in BCR-ABL-mediated induction of Survivin were studied using inhibitors specific for each signaling cascade. The effect of Survivin on the proliferation and viability of IM-sensitive and IM-resistant CML cells was investigated after silencing Survivin expression with small interfering RNAs. Results: BCR-ABL tyrosine kinase activity induced an over-expression of Survivin in both human and murine hematopoietic cell lines. This over-expression was both at the transcriptional and the translational level and required the JAK2/STAT3 pathway. Survivin silencing by siRNA increased IM cytotoxicity in IM-sensitive cells but failed to restore IM efficacy in IM-resistant cells. However, Survivin silencing sensitized CML cells to the cytotoxic effect of hydroxyurea and enhanced the efficacy of this compound on three different murine cell lines are insensitive to IM because of point mutations in the BCR-ABL kinase domain (Ba/F3p210Y253F, Ba/F3p210D276G and Ba/F3p210T315I). Conclusions: Reduction of Survivin expression improves the efficacy of IM and increases the sensitivity of IM-resistant CML cells to hydroxyurea. Survivin may represent an attractive therapeutic target for both IM-sensitive and IM-resistant CML patients. No significant financial relationships to disclose.

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