The Kinase Inhibitor TKI258 Is Active Against the Novel CUX1-FGFR1 Fusion Detected In a Patient with T-Lymphoblastic Leukemia/Lymphoma and t(7;8)(q22;p11)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2715-2715
Author(s):  
Bartosz Wasag ◽  
Els Lierman ◽  
Peter Meeus ◽  
Jan Cools ◽  
Peter Vandenberghe
Keyword(s):  
Cell Line ◽  
Western Blotting ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Antiproliferative Effect ◽  
Fusion Partner ◽  
The Novel

Abstract Abstract 2715 The 8p11 myeloproliferative syndrome (EMS) is an aggressive, atypical stem cell disorder associated with chromosome translocations that constitutively activate FGFR1 by fusion to diverse partner genes. Here, we describe a case with a clinical and hematological diagnosis of T-lymphoblastic leukemia/lymphoma and with a t(7;8)(q22;p11) on cytogenetic analysis. We identified the fusion partner involved and characterized this translocation functionally in vitro using the interleukin 3 (IL3) dependent Ba/F3 cell line. The translocation was analyzed in more detail by FISH using FGFR1 flanking probes. We could confirm the 8p11 breakpoint and 7q as the partner chromosome. Using 5'-RACE CUX1 (7q22) was identified as the fusion partner of FGFR1 in this patient with T-lymphoblastic leukemia/lymphoma. CUX1 is a homeobox family DNA binding protein not previously described as a fusion partner in hematological malignancies. To evaluate the transforming potential of this novel fusion, the CUX1-FGFR1 fusion was cloned and used to transform Ba/F3 cells. CUX1-FGFR1 expressing Ba/F3 cells displayed IL3 independent proliferation thus demonstrating the oncogenic character of this fusion protein. Western blotting of the transformed Ba/F3 cells showed activation of FGFR1 as well as its downstream target STAT5. Treatment of the CUX1-FGFR1 expressing Ba/F3 cells with the kinase inhibitors PKC412 and TKI258 significantly inhibited cell growth with an IC50 of 483 and 489 nM respectively. With western blotting a direct effect of both inhibitors on FGFR1 kinase activity as well as on different downstream effectors was proven. Furthermore using an annexinV/propidium iodide-based apoptosis assay, we could show that PKC412 and TKI258 both induced apoptosis followed by cell death in inhibitor treated CUX1-FGFR1 transformed Ba/F3 cells. The antiproliferative effect of the inhibitors could be rescued by addition of IL3 for the TKI258 treated but not for PKC412 treated CUX1-FGFR1 expressing cells. This observation indicates a selective action of TKI258 on FGFR1 signaling at the concentrations used. In contrast, for PKC412 non-specific cytotoxicity is also contributing to the antiproliferative effect. In summary, we identified a novel CUX1-FGFR1 fusion in a case with EMS and a novel t(7;8)(q22;p11), and demonstrated the oncogenic potential of CUX1-FGFR1 in the Ba/F3 cell line. This new fusion partner CUX1 contains a potential coiled coil domain that can explain the observed constitutive FGFR1 activation, as has been elaborately demonstrated for other oncogenic kinase fusions. The in vitro data presented here using the inhibitor TKI258 support the use of this compound for the treatment of the novel CUX1-FGFR1 fusion as well as other constitutively active FGFR1 fusion proteins. Disclosures: No relevant conflicts of interest to declare.

Transient Responses to Notch and Tlx1/Hox11 Inhibition In T-Cell Acute Lymphoblastic Leukemia/Lymphoma.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1026-1026
Author(s):  
Erica A. Lehotzky ◽  
Mark Y. Chiang
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cell Line ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Ectopic Expression ◽  
Leukemia Cells ◽  
Inhibitory Effects ◽  
Notch Inhibition

Abstract Abstract 1026 Despite numerous advances in the past few decades, treatment of acute lymphoblastic leukemia/lymphoma (ALL) remains a common and considerable challenge. Further efforts to define the molecular lesions that drive ALL are needed to improve clinical management. The Hox subfamily of T-cell ALL (T-ALL) represents 30–40% of pediatric and adult cases. TLX1/HOX11 is the prototypical member of the Hox group. To generate a resource for developing targeted therapies for Hox T-ALLs, we developed a doxycycline-regulated mouse model of Tlx1-initiated T-ALL. Dysregulated thymic expression of Tlx1 induces T-ALL after ∼5-7 months with penetrance of 15–60%. The lymphoblasts are arrested at the early CD4+/CD8+/CD24hi stage of T-cell development, similar to human T-ALLs of the TLX1 subtype. Spontaneous activation of the Notch1 oncogene occurred in the tumors. In about two-thirds of samples, Notch was activated through acquired mutations in the heterodimerization and PEST domains that resemble the Notch1 mutations found in human patients. Inhibition of Notch signaling with g-secretase inhibitors completely abrogated cell line growth and induced apoptosis. Notch inhibition also transiently delayed leukemia progression by ∼17 days in vivo. In contrast, suppression of Tlx1 expression had more moderate inhibitory effects on cell line growth in vitro. However, suppression of Tlx1 expression in transgenic mice transiently delayed leukemia progression by ∼11 days. Tlx1 suppression had the strongest inhibitory effects on expression of CCR7 and lymph node size. These effects were fully reversed with ectopic expression of Tlx1. These data show that Tlx1 can convert normal thymocytes into leukemia cells, but the leukemia cells are not fully dependent on continued Tlx1 expression. The leukemia cells recruit secondary factors and pathways such as Notch and c-Myc to sustain growth and survival. Our study highlights a strong resiliency of T-ALL cells to both Tlx1 and Notch inhibition. Our study has important implications for targeting these pathways for the treatment of T-ALL. Disclosures: No relevant conflicts of interest to declare.

The Small Molecule Inhibitor of VLA4 TBC3486 Sensitizes Resistant ALL to Chemotherapy

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1500-1500 ◽  
Author(s):  
Yao-Te Hsieh ◽  
Eun Ji Gang ◽  
Halvard Bonig ◽  
Ronald J Biediger ◽  
Peter Vanderslice ◽  
...  
Keyword(s):  
Cell Viability ◽  
Small Molecule ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Chemotherapy Resistance ◽  
Leukemia Cell ◽  
The Novel ◽  
Small Molecule Inhibition

Abstract Abstract 1500 Significant progress notwithstanding, drug resistant acute lymphoblastic leukemia (ALL) remains a therapeutic challenge, as well as acute and long-term off-target toxicity of anti-ALL therapies can be dose-limiting or debilitating. Therefore, the development of more targeted therapies is desirable. We recently provided evidence that chemotherapy resistance of ALL cells can be partly overcome by interfering with the function of VLA4, the alpha4beta1 integrin, in vivo. In those studies, we used the anti-functional antibody Natalizumab. We extended our studies to an alternative VLA4 inhibitor, the novel non-peptidic small molecule TBC3486. Previous in vitro assays and molecular modeling studies indicate that TBC3486 behaves as a ligand mimetic, competing with VCAM-1 for the MIDAS site of VLA-4. As such, the compound has been shown to be efficacious in VLA-4 dependent models of inflammatory and autoimmune disease. The potential usefulness of this novel inhibitor in leukemia treatment was tested in our established in vitro and in vivo assays. LAX7R cells, primary pre-B-ALL with a normal karyotype from a patient with an early relapse, were used throughout for the studies reported here. LAX7R cells were treated with 25μM TBC3486 or THI0012 control, the inactive enantiomer of TBC3486, and seeded onto plates coated with human VCAM-1. Adhesion, scored after 2 days, was significantly inhibited by TBC3486 compared to control treated cells (7.9%±4.0 vs 95.4%±8.0; p=0.003). Proliferation rate and cell viability were unaffected by the treatments. In a co-culture system of LAX7R cells with OP9 stroma cells, which we use as an in vitro model of stroma-mediated chemotherapy resistance, we assessed differential effects of VDL (Vincristine, Dexamethasone, L-Asparaginase) on leukemia cell survival in the presence or absence of TBC3486. Stromal adhesion significantly protected LAX7R cells against VDL chemotherapy; this effect was significantly attenuated by TBC3486 compared to the control as determined by Trypan blue exclusion of dead cells (Cell viability of 39.9%±5.1 vs. 57.2±1.8; p=0.02). After these encouraging observations, we next evaluated the benefit of TBC3486 on leukemia progression in a xenotransplant assay. LAX7R cells were lentivirally labelled with luciferase for in vivo tracking and injected into NOD/SCID hosts. Three days after leukemia cell transfer, mice received either TBC3486 or THI0012 (control) (10mg/kg/d) daily for 2 weeks (intraperitoneally), with or without VDL chemotherapy. This experiment is in progress, but already survival of leukemia-bearing mice was significantly prolonged, from a median survival time (MST) for control mice of 33 days post-leukemia injection to a MST of 47 days post-leukemia injection for TBC3486 treated mice (p=0.02). Similarly, bioluminescence imaging revealed a marked delay of leukemia cell dissemination (p<0.0001). Taken together, our data demonstrate that small molecule inhibition of VLA4 using the novel TBC3486 is a suitable approach for targeting of chemotherapy-resistant leukemia. Further studies are warranted to understand and evaluate preclinically adjuvant small molecule inhibition of integrins to overcome relapse of ALL. Disclosures: No relevant conflicts of interest to declare.

Novel Tyrosine Kinase Inhibitors Trigger Drug Sensitivity of Primary CLL Cells by Controlling Glucosylation of Ceramides

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3905-3905
Author(s):  
Janine Schwamb ◽  
Valeska Feldhaus ◽  
Michael Baumann ◽  
Michaela Patz ◽  
Susanne Brodesser ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Treatment Options ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Published Data ◽  
The Novel ◽  
Apoptosis Resistance

Abstract Abstract 3905 Background: Apoptosis resistance of chronic lymphocytic leukemia (CLL) cells is mediated by several pro-survival stimuli. In particular, engagement of the B-cell receptor (BCR), CD40-CD40 ligand (CD40L) interaction or stimulation by interleukin-(IL)-4 were identified as major factors to regulate chemoresistance. Sphingolipids are known to be involved in several metabolic pathways involved in chemoresitance. Therefore, we focused on ceramide as pro-apoptotic molecule and its counterpart glucosylceramide, which rather contributes to proliferation and survival. Methods and Results: Applying liquid chromatography electrospray ionization tandem mass spectrometry, we revealed a significant decrease of pro-apoptotic ceramide in BCR/IL-4/CD40L-stimulated primary CLL cells compared to untreated controls (p=0.0258, p=0.0478, p=0.0114). Anti-apoptotic glucosylceramide levels were significantly increased after BCR cross-linking (p=0.0435) while other stimuli caused no relevant change in glucosylceramide expression. We identified BCR engagement to catalyze the crucial modification of ceramide to glucosylceramide via the enzyme UDP-glucose ceramide glucosyltransferase (UGCG) (p=0.0001). Besides specific UGCG inhibitors, we could show for the first time that IgM-mediated UGCG expression was significantly inhibited by the novel and highly effective PI3Kδ and BTK inhibitors CAL-101 and PCI-32765, which were able to revert IgM-induced apoptosis resistance of CLL cells. Recently published data revealed sphingolipids to be essential for mediation of apoptosis via mitochondria. Therefore, we chose ABT-737 – a well-known and also mitochondria-targeting drug – as candidate partner for PI3Kδ and BTK inhibition. When combining each tyrosine kinase inhibitor with ABT-737, a synergistic apoptotic effect could be documented, even under protection by BCR stimulation. Conclusion: In summary, we could demonstrate that sphingolipids are critically involved in CLL pathogenesis. UGCG could be identified as drugable target by the novel kinase inhibitors CAL-101 and PCI-32765 resulting in even synergistic apoptosis following additional application of ABT-737. Sphingolipids seem to offer further targets providing novel treatment options in CLL. C.M.W. and L.P.F. contributed equally to this work. Disclosures: No relevant conflicts of interest to declare.

Flt3 Is Required for MLL-ENL-Induced Leukemogenesis In Vivo, but Not for LSC Function in Vitro

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2450-2450
Author(s):  
Kenjiro Kamezaki ◽  
Hans-Willem Snoeck
Keyword(s):  
Kinase Inhibitors ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Tyrosine Kinase Receptor ◽  
Mouse Bone Marrow ◽  
Retroviral Transduction ◽  
Significant Difference

Abstract Abstract 2450 Flt3 is a type III tyrosine kinase receptor expressed on hematopoietic multipotential progenitors and more downstream progenitor cells in the mouse bone marrow (BM).Flt3 is one of the most frequently mutated genes in acute myeloblastic leukemia (AML) patients, leading to constitutive activation. Several Flt3 kinase inhibitors are in clinical development in AML with Flt3 mutations, although disappointing results suggest that Flt3-inhibitors as monotherapy are unlikely to become reality. Wild type (wt) Flt3, which differs in its signaling characteristics and downstream targets from mutated Flt3, is highly expressed in particular in AML and ALL (acute lymphoblastic leukemia) with MLL (Mixed Lineage Leukemia) rearrangements. These observations suggest a role for wt Flt3 in pathogenesis as well, although little experimental or clinical evidence has been published yet supporting this notion. To our knowledge, there is only one report suggesting that wt Flt3 is required for the development of leukemia in a murine model of c-cbl mutation-induced leukemogenesis (Rathinam C, et al. Cancer Cell 2010). For this reason, we focused on the role of wt Flt3 in leukemogenesis and using leukemia mouse models established by retroviral transduction of MLL-ENL, MLL-AF9 and AML1-ETO9a. Mice transplanted with MLL-ENL-transduced Flt3−/− BM cells showed a higher survival rate (wt 61.3%, n=34 vs Flt3−/− 96.8%, n=31, median follow-up period; 255 days, p=0.038). No difference in survival was observed in the MLL-AF9 and AML1-ETO9a models however. Surprisingly, MLL-ENL-transduced Flt3−/− BM cells showed no significant difference in serial replating capacity of BM cells in methylcellulose colony forming assays in vitro compared to wt BM cells, and putative lin-kit+ leukemic stem cells (LSCs) did not express Flt3. In vivo, however, putative MLL-ENL LSCs, but not MLL-AF9 LSCs expressed Flt3. Furthermore, in non- and pre-leukemic MLL-ENL-transduced mice BM cells the frequency of lin-kit+ putative LSCs among MLL-ENL positive populations was decreased in Flt3−/− group (wt 1.468% vs Flt3−/− 0.466%, p=0.031). Flt3+ lin-kit+ populations were increased in MLL-ENL transduced leukemic wt mice compared to non-leukemic mice (leukemic mice 26.7% vs non-leukemic mice 9.9%, p=0.028). These results suggest that Flt3 signaling is required for MLL-ENL-induced leukemogenesis and maintenance of LSCs in vivo, but not in vitro. Importantly, the data suggest that the in vitro replatable leukemia stem cell (LSC) paradigm might not reflect LSC function or phenotype in vivo. Disclosures: No relevant conflicts of interest to declare.

MLL Rearrangement and Age At Diagnosis Are Strongly Associated with High Level Surface FLT3 Expression and Ex Vivo Sensitivity to FLT3 Inhibition: A Prospective Analysis of 54 Consecutive Infants with ALL Enrolled in Children's Oncology Group (COG) Trial AALL0631

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 568-568
Author(s):  
Allison J. Kaeding ◽  
Daniel Magoon ◽  
Yarden Fraiman ◽  
Tamekia Jones ◽  
Nyla A. Heerema ◽  
...  
Keyword(s):  
Protein Expression ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Predictive Biomarkers ◽  
Age At Diagnosis ◽  
Fusion Partner ◽  
Childhood All ◽  
Flt3 Inhibitor ◽  
Flt3 Expression

Abstract Abstract 568 Background: Infant acute lymphoblastic leukemia (ALL) is clinically and biologically distinct from ALL in older children. About 80% of infant ALL cases harbor MLL rearrangements (MLLr). MLLr infant ALL is an aggressive disease with poor prognosis, particularly in cases diagnosed at <90 days of age. Infant cases with wild type MLL (MLLwt) are clinically similar to childhood ALL. Retrospective studies of selected banked specimens have suggested that MLLr infant ALL cases express higher levels of FLT3 receptor tyrosine kinase mRNA and protein, and show enhanced in vitro FLT3 inhibitor-induced cytotoxicity compared to MLLwt childhood ALL, leading to an ongoing COG trial to determine if adding lestaurtinib to chemotherapy improves outcome for MLLr infant ALL (AALL0631). Methods: We prospectively characterized 54 consecutive diagnostic leukemia specimens from infants with ALL enrolled on AALL0631 with respect to: 1) Quantitative surface FLT3 protein expression (s-FLT3) using FACS CD135 mean fluorescence index (MFI); 2) FLT3 inhibitor (lestaurtinib: 0 to 100 nM) in vitro sensitivity using 48 hour WST-1 cytotoxicity assays; samples defined as “sensitive” if 100 nM produced ≥40% inhibition of cell viability compared to vehicle controls; 3) Identification and quantification of putative leukemia stem cell (LSC) subpopulation by flow cytometric immunophenotyping (% CD34+, CD38− viable cells). Studies were performed blinded to clinical information and MLL status; data were later correlated with age at diagnosis (<90 days vs. ≥90 days) and MLL-rearrangement status/fusion partner (cytogenetics/FISH). Results: Of 54 cases, 42 (78%) are MLLr and 12 (22%) are MLLwt. For MLLr cases, fusion partners are AF4 (n=13), ENL (n=17), AF9 (n=3), AF1p (n=2), and other (n=7). Of MLLr cases, 10 (24%) were <90d and 32 (76%) were ≥90 days. All MLLwt cases were ≥90 days.We analyzed s-FLT3 according to MLL genotype, MLL fusion partner and age, and found significantly higher s-FLT3 in MLLr than MLLwt cases (mean MFI 34.2 vs. 11.6, p=0.03). According to MLL fusion partner, we found the highest s-FLT3 in AF9, lowest in AF4, and intermediate in ENL and AF1p. The AF9 vs. AF4 comparison was significant (p=0.006). According to age in the MLLr cohort, we found strikingly higher s-FLT3 in infants diagnosed at <90days compared to those >90days (mean MFI 62.6 vs. 22.0, p<0.0001). We analyzed FLT3 inhibitor sensitivity according to MLL genotype and s-FLT3. All 42 of the MLLr samples were evaluable, with 35 (83%) sensitive and 7 (17%) resistant; 11 of 12 MLLwt samples were evaluable (1 had excessively poor viability), with 6 (55%) sensitive and 5 (45%) resistant. We thus found a significant association between MLLr and FLT3 inhibitor sensitivity (Chi square 4.125, p=0.042). In the s-FLT3 analysis, the sensitive samples trended towards higher s-FLT3 than the resistant samples (mean MFI 31.3 vs. 16.7, p=0.17). Finally, we compared s-FLT3 in the putative LSC-like subpopulation fraction (if present) with the “bulk” leukemia population. Of the 54 samples analyzed, 35 (65%) had a clearly identifiable LSC-like population (i.e., comprising >0.5% of the total viable leukemia population). Within these 35 samples, s-FLT3 was significantly higher in the LSC-like fraction than in the bulk leukemia population (mean MFI 40.4 vs. 31.2, p=0.05). Conclusions: We have for the first time prospectively compared FLT3 expression and FLT3 inhibitor sensitivity in unselected representative cohorts of MLLr and MLLwt infants, confirming that MLLr cases express significantly higher levels of surface FLT3 protein and are more sensitive to the cytotoxic effects of FLT3 inhibition. Novel findings include higher surface FLT3 protein expression in MLL-AF9 cases relative to MLL-AF4 cases, and the strikingly higher surface FLT3 protein expression in MLLr cases in the youngest infants. An intriguing finding is the presence of phenotypically-defined subpopulations of “LSC-like” cells in the majority of cases, and that this LSC-like population expresses significantly higher levels of FLT3 protein than the “bulk” population. These findings provide further evidence that FLT3 overexpression plays a role in MLLr leukemogenesis. If these findings correlate with clinical responses in COG AALL0631, they may prove to be useful predictive biomarkers in selecting patients for whom FLT3-targeted therapy is most appropriate. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Phosphoproteomic Landscaping Unveils Constitutive cKIT Activation in Human Erythroblasts from Polycythemia Vera (PV) Patients

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 399-399
Author(s):  
Lilian Varricchio ◽  
Giulia Federici ◽  
Francesca Masiello ◽  
Fabrizio Martelli ◽  
Mario Falchi ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Progenitor Cells ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Cell Cycle Control ◽  
Major Change ◽  
Fold Increase ◽  
Mapk Signaling

Abstract PV is characterized by the gain-of-function V617F mutation in JAK2, the gene encoding the first signaling element of the cytokine receptor superfamily. Progenitor cells from PV are more sensitive in vitro to Imatinib, which also inhibits the SCF receptor cKIT, than those from normal sources (Gaikwad, Exp Hemat 2007;35:931) and clinical trials with similar tyrosine kinase inhibitors have been reported to have some efficacy in PV (Nussenzveig, Int J Hematol 2009;90:58; Silver, Leuk Res 2012;36:156). These data led us to examine the mechanism by which this tyrosine kinase inhibitor affects erythropoiesis in PV. We observed that in cultures containing SCF PV progenitor cells generated similar numbers of erythroid cells (Ery) as those from adult (AB) and cord (CB) blood by day 10 [fold increase (FI) ~1-2] but by day 13 PV cells generated significantly greater numbers of Ery than AB and CB [PV FI=11±0.2, p=0.021 vs AB; CB FI=6.2±1.9, p=0.025 vs PV and 0.0055 vs AB; AB FI=2.6±0.5]. Since by day 10 progenitor cells were no longer detected, we hypothesized that increases in Ery at day 13 reflect intrinsically greater Ery proliferation potentials. To test this hypothesis, we compared the phosphoproteomic landscaping of day 10 Ery from 3 PV, 3 AB and 3 CB by Reverse Phase Protein Array (RPPA) using as target >160 signaling events (data are at http://capmm.gmu.edu/data). Overall, 40 proteins were statistically different between PV and AB and 30 proteins were statistically different between CB and AB. Pathway analyses of significant hits identified that PV and CB Ery differ from the AB ones in the activation states of 1-2 proteins involved in stemness and cell cycle control inferring that there is no major change in their cycling or differentiation state. By contrast, the 3 populations showed numerous differences in cKIT signaling. PV Ery differed from AB cells by expressing greater levels of cKITY719 and cKIT703, which were reduced to barely detectable levels by the pan-JAK inhibitor Ruxolitinib, and of elements of PI3K (eNOS/NosIII, PDK1 and PKCd) and MAPK (pMARCKS, MSK1, AMPKα1 and β1 and p38 MAPK) signaling downstream, respectively, to cKITY719 and cKIT703. PV Ery expressed also greater levels of JAK2Y1007/1008 and of its downstream target STAT3Y705. CB Ery showed lower levels of cKIT, cKITY703, cKITY719 and CD63, a member of tetraspanin superfamily that binds cKITY719 switching its intracellular fate from recycling to lysosome degradation, greater phosphorylation of proteins of MAPK (pMARCKS, MSK1, PTEN and Src) and PI3K (PKCd, mTOR, p70S6K and panPKC/βII) signaling than AB Ery. These results were stoichiometrically validated by WB and indicate that PV Ery express greater degrees of cKIT activation than AB Ery suggesting that greater response to SCF might account for their greater amplification in culture. This hypothesis was tested by RPPA analyses (and stoichiometric validation by WB) of Ery from PV, AB and CB growth factor deprived (GFD) for 4h and then stimulated with SCF for 15' and 2h. GFD altered the activation state of 25 proteins (22 de-activated and 3 activated) in PV, of 12 proteins (10 de-activated and 2 activated) in AB and 8 proteins (4 de-activated and 4 activated) in CB. SCF altered the activation state of 36 proteins in PV (18 activated and 18 de-activated), 23 proteins in CB (all activations) and 6 proteins in AB (all activations). In PV and CB Ery, GFD decreased cKITY719 and cKITY703 and the activation state of their downstream targets JAK2Y1007/1008, MAPKs and mTOR while SCF increased the stoichiometric levels of cKITY719 and cKITY703 and the activation of mTOR. SCF also increased cKITY703 and cKITY719 but did not activate mTOR in Ery from AB. In agreement with the hypothesis that Ery from PV and CB respond more readily to SCF than those from AB, SCF induced greater cell-surface cKIT down-modulation (by flow cytometry) and lower intra-cytoplasmic cKIT/CD63 association (by confocal microscopy and WB) in PV and CB Ery than in AB Ery. Screening of 97 inhibitors against targets analysed by RPPA which are approved for clinical use by FDA revealed that growth of PV Ery was more sensitive than that of AB only to JAK and cKIT inhibitors. In addition, shRNA-CD63 reduced the growth of PV Ery (FI=0.9 vs 1.3 p=0.012) while increased by 2-fold (p=0.02) that of AB Ery. These results provide the first phosphoproteomic landscaping of cKIT signalling in Ery from PV and normal sources and confirm that cKIT is an important therapeutic target for PV. Disclosures No relevant conflicts of interest to declare.

Deciphering the Critical Pathways of Mutant N-RAS in AML Using Small Molecule Inhibitors.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2455-2455
Author(s):  
Atsushi Nonami ◽  
Martin Sattler ◽  
Ellen L. Weisberg ◽  
Liu Qingsong ◽  
Jianming Zhang ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Small Molecules ◽  
Cell Line ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Leukemia Cell ◽  
Small Gtpase ◽  
Ras Signaling ◽  
Chemical Screen

Abstract Abstract 2455 Activating mutations in the small GTPase N-RAS occur in about 10% of acute myeloid leukemia (AML) cases. Active N-RAS is thought to drive the disease and is therefore a potential target for drug development. There have been numerous unsuccessful efforts to target RAS itself with small molecules, and blocking post-translational modifications of RAS proteins, such as through inhibition of farnesyl transferase, has similarly not proven useful. In addition, efficacy of targeting critical downstream effectors has been limited by the complexity of RAS signaling, such as redundancy of signaling pathways and feedback mechanisms. While targeting RAS is challenging, it was our hypothesis that inhibiting the right combination of downstream pathways in a particular lineage with small molecules could be effective. Initially, we created a Ba/F3 cell line that was completely dependent on oncogenic N-RAS-G12D for growth and survival. Growth was suppressed >99% by shRNA for N-RAS, but could be rescued entirely by interleukin-3 (IL-3), which does not require N-RAS signaling in these cells. Using this cell line, we performed a high-throughput chemical screen with a large library of multi-targeted kinase inhibitors. The lead compound (NRAS1) showed a 70-fold difference in the EC50 for growth inhibition between BaF3-NRAS G12D cells cultured in the absence (0.01μM) or presence (0.77μM) of IL-3. Importantly, this compound showed selectivity towards several leukemia cell lines that were shown to be dependent on mutant N-RAS by shRNA compared to cells expressing wild-type N-RAS (p=0.02). Also, in a xenotransplant model using NRAS-G12D+ OCI-AML3 cells, this compound significantly reduced tumor burden (P=0.005) and prolonged survival (P=0.002) compared to controls. Next, we sought to identify the targets of NRAS1, Interestingly, the compound did not suppress MEK or ERK, which are classical targets of RAS signaling in epithelial cells. NRAS1 profoundly reduced AKT and RPS6 phosphorylation. Kinase selectivity profiling of this compound (1μM) in OCI-AML3 cells (EC50: 0.3μM) identified 13 major binding partners with more than 85% efficacy. The targets consisted mainly of SRC family proteins (SRC, FGR, and LYN etc.) and MAPK family proteins (MAP4K2, 3, 5, and p38 etc.) and others (ZAK and BTK etc), but not MEK and ERK, and AKT was not detected in this assay. In preliminary studies, most of these target kinases were knocked-down by shRNA and, as expected, no single kinase was found to be responsible for mediating growth inhibition. Using a phospho-antibody microarray, the most significantly de-phosphorylated kinases were p38, AKT and SRC, which supports our preliminary findings. To validate the significance of these results, we treated Ba/F3-N-RAS cells with combinations of kinase inhibitors. Combining the AKT inhibitor MK2206 and Dasatinib (SRC family inhibitor) revealed marked synergy, while neither had activity individually. Also, the combination of MK2206 and a cleaner SRC family inhibitor, AZD0530, also synergized, although to lesser extent. In both examples, however, the inhibition of N-RAS transformed cells by NRAS1 proved superior, suggesting that one or more additional targets are required for inhibition of NRAS signaling. To identify additional critical targets of our compound we generated several derivatives with different potency. In particular, one less potent analog of NRAS1 (analog 6, 1% EC50 of original compound) showed a loss of binding activity towards the MAP4K family of proteins, especially MAP4K2. Observed synergy between the selective MAP4K2 inhibitor NG25 and selective inhibitors of MK2206 and Dasatinib in Ba/F3-NRAS G12D cells further points toward MAP4K2 as being of additional significance for oncogenic RAS signaling. Together with the previous data, we propose AKT and MAP4K2 as critical targets of NRAS1. In conclusion, we have identified a novel and selective kinase inhibitor of the N-RAS signaling pathway by chemical screen using Ba/F3-N-RAS G12D cells. By combination of signaling study, kinase selectivity profiling and phosphoproteomics, the main functional targets were found to be AKT, and MAP4K2, and additional functional targets will be elucidated. Our approach also could be applied for other type of oncogenes, and it could help to find therapeutic compound and also help to decipher signaling mechanisms of the oncogenes which are thus far undruggable. Disclosures: No relevant conflicts of interest to declare.

Clarithromycin Targeting Autophagy Potentiates Tyrosine Kinase Inhibitor-Induced Cell Death in Resistant Chronic Myeloid Leukemia (CML) Patients

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4450-4450
Author(s):  
A. M. Carella ◽  
Gioacchino Catania ◽  
G. Beltrami ◽  
G. Pica
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitor ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Blast Crisis ◽  
Cytogenetic Response ◽  
Ifn Alpha

Abstract Abstract 4450 M-TOR is a key regulator of autophagy. Rapamycin and clarithromycin (structurally similar to rapamycin), have been demonstrated to have in vitro activity in blocking autophagy. In four patients with advanced CML, remarkable response to the combination of clarithromycin and a tyrosine kinase inhibitor was observed. Here we present the results achieved by the combination. A 43-year-old woman was diagnosed with high-risk Sokal CML in February 2000. She was treated with IFN-alpha and imatinib (400 mg/day) with persistence of 100% Ph-positive metaphases. In March 2006, WBC was no longer controlled and she was treated with nilotinib. Complete hematologic response (CHR) was achieved by the end of April 2006, but there was no cytogenetic response (CyR). She was given dasatinib (70 mg b.i.d.) without complete cytogenetic response (CCyR) and after 7 months the bcr-abl/abl ratio was 6.1% in March 2011. At that time, the patient had an infection (otits/pharyngitis) sensitive to clarithromycin, which was added to dasatinib at a dose of 500 mg b.i.d. April 2011 there was a surprising reduction in the transcript to 0.5%. As of June 2011, the value was 0.05%, and the patient continues to receive clarithromycin (500 mg/day) and dasatinib (100 mg/day). Nowadays (August 1), the patient is in CHR, CCyR and major molecular remission (MMR) (bcr-abl/abl ratio 0.001%). The patient stopped clarithromycin and he is continuing on dasatinib. A 53-year-old man was diagnosed with de novo lymphoid blast crisis CML in August 2010; bcr-abl/abl ratio was 95.2%. He had a sibling donor. In October 2010 bcr-abl/abl ratio was reduced to 0.2% after chemo + imatinib. In November 2010, bcr-abl/abl ratio was 22% and he was treated with dasatinib (70 mg b.i.d.) with WBC control and a small reduction of bcr-abl/abl ratio (18% in February 2011). Soon thereafter, he underwent allogeneic transplant. Two months after transplant (May 2011) the disease progressed and bcr-abl/abl value had increased to 47%. He was restarted on dasatinib (100 mg/day) but the transcript increased in 4 weeks to 143%. Because of our previous experience, we added clarithromycin to dasatinib on June 2, 2011. Two weeks later, bcr-abl/abl value was reduced to 3.2%, and to 1.5% after another week. We stopped clarithromycin and three weeks later under dasatinib alone the transcript increased to 20%. From one week we added newly clarithromycin to dasatinib. A 68 year old man was diagnosed with CML in October 1999. A CCyR was achieved after autografting and soon after IFN-alpha was given as maintenance. In October 2000 the patient relapsed. A second CCyR was achieved in December 2001 after imatinib (400 mg/day), which lasted for six years. In October 2006 bcr-abl/abl ratio was 4.5%. He was treated with dasatinib (70 mg. b.i.d.) with WBC control but with no CyR. In March 2011, bcr-abl/abl ratio was 42.5%. Nilotinib (600 mg. b.i.d.) was begun with no change in bcr-abl/abl ratio after 2 months. In June 2011, clarithromycin (500 mg. b.i.d.) was added; 3 weeks later, the bcr-abl/abl ratio had decreased to 17% and two weeks later (July 13, 2011) to 4%. On July 28, bcr-abl/abl is 0,00022%. A 70 year old woman was diagnosed with CML in November 1998. She was treated with IFN-alpha but only partial CyR was achieved. In January 2001, 100% Ph-positive metaphases were found in BM. She was begun on imatinib (400 mg/day) but the karyotype did not change. In May 2005 she was started on nilotinib (600 mg/daily) since bcr-abl/abl ratio was 26.5%. Blood counts were controlled but there was no change in cytogenetics. In August 2010 WBC increased to 100×103/l. Dasatinib (70 mg. b.i.d.) was begun. Because blood count control was inadequate, hydroxyurea was added. In December 2010, bcr-abl/abl ratio had increased to 140%, and E255V mutation was found. In May 2011, clarithromycin (500 mg. b.i.d.) was added. In 2 weeks, the WBC had decreased from 76×103/l to 10×103/l and bcr-abl/abl ratio was 30% (June 4, 2011). One month later (July 4, 2011) bcr-abl/abl ratio was 3% and the mutation was no longer found in bone marrow. In the last evaluation (July 13, 2011) bcr-abl/abl ratio was 0.00096%. The patient stopped clarithromycin and she is on dasatinib alone. In conclusion, no patients have gone off study for toxicity. In no case we observed grade 3–4 myelosuppression. The remarkable responses obtained in these 4 patients support the hypothesis that inhibition of autophagy may make CML cells sensitive to killing by tyrosine kinase inhibitors. Disclosures: No relevant conflicts of interest to declare.

Integrated Analysis Of CRLF2 Signaling In Acute Lymphoblastic Leukemia Identifies Polo-Like Kinase 1 As a Therapeutic Target

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2667-2667
Author(s):  
Tai-Chung Huang ◽  
Shubhada Shashikant Bharne ◽  
Jevon Cutler ◽  
Jun Zhong ◽  
David M. Weinstock ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Ex Vivo ◽  
Lymphoblastic Leukemia ◽  
Total Body Weight ◽  
Inhibitor Screening ◽  
Total Body ◽  
Bi 2536

Abstract Introduction Acute lymphoblastic leukemia is the most common cancer in children, and the five-year survival rate among patients older than 20 years old is still poor. Recently, Cytokine Receptor-Like Factor 2 (CRLF2) was identified as a prognostic factor in 15% of precursor B cell-acute lymphoblastic leukemia (B-ALL) that lack typical gene rearrangements (Yoda et al., Proc Natl Acad Sci U S A. 2010). In addition, several independent studies also demonstrated that CRLF2 acts as an oncoprotein (Mullighan et al., Nat Genet. 2009; Russell et al., Blood. 2009; Chapiro et al., Leukemia. 2010). The Children’s Oncology Group also found that high expression of CRLF2 confers an adverse prognosis in high-risk B-ALL patients (Chen et al., Blood. 2012). The mechanism of CRLF2-associated leukemogenesis, however, is still not clear. In CRLF2-rearranged B-ALL cases, CRLF2 is overexpressed because of genetic rearrangements. Interestingly, one-half of cases co-harbor an activating mutation in exon 16 of JAK2, most commonly involving R683 or I682. In a murine progenitor B cell line, BaF3, it has been shown that the combination of CRLF2 overexpression and JAK2 R683G confers IL-3-independent growth. This illustrates that CRLF2 overexpression could synergize with JAK2 R683G to trigger leukemic proliferation. In our previous phosphoproteomics analysis of BaF3 cells expressing CRLF and JAK2 R683G, the combination upregulated phosphorylation of Protein phosphatase 1, regulatory subunit 12A (Ppp1r12a) (van Bodegom et al., Blood. 2012), which is a negative regulator of Polo-like kinase 1 (Plk1) (Yamashiro et al., Dev Cell. 2008). In this study, we aimed to focus on novel approaches to target CRLF2 signaling in B-ALL. Methods To identify effective kinase inhibitors against leukemic CRLF2 signaling, we subjected BaF3 cells with CRLF2 overexpression and JAK2 R683G (BJC cells) to a kinase inhibitor screening system that consisted of 73 kinase inhibitors and other small molecules. An in vitro cell proliferation assay was coupled to observe growth inhibition at 37°C for 3 days (Tyner et al., Cancer Res. 2012). To validate ex vivo efficacy of the Plk1 inhibitor (BI-2536), which scored in the screen, we injected BJC cells with/without pre-treatment for 24 hours at 100 nM into the NOD-scid IL2Rγnull (NSG) mice and compared spleen/liver sizes 15 days after injection. To compare in vitro efficacy of Plk1 inhibitor in CRLF2-rearranged B-ALL xenografts, we used human hTERT-transformed bone marrow stromal cells as a feeder layer and green fluorescent dye (488 nm) viability staining for flow cytometry analysis 48 hours after treatment. To investigate the downstream effect of dysregulated Plk1 in BJC cells, morphology of centrosomes was examined using anti-g-tubulin immunofluorescence staining. Results The kinase inhibitor screening showed that the Plk1 inhibitor BI-2536 is particularly potent against BJC cells with an IC50 of 11 nM. To validate this screening result and test the efficacy of BI-2536 ex vivo, NSG mouse transplantation experiments showed that BJC cells that were pretreated with BI-2536 had significantly smaller spleen (0.47% vs. 1.45% of total body weight, p<0.01) and liver (5.53% vs. 7.62% of total body weight, p<0.01) sizes compared with BJC cells without treatment. The efficacy of BI-2536 against human CRLF2-rearranged B-ALL xenografts was evaluated in vitro. In comparison with vehicle control, the viability of CRLF2-rearranged B-ALL cells treated with BI-2536 was lower (48.8% of vehicle) than a wild-type CRLF2 B-ALL with BI-2536 (105.3% of vehicle). Because Plk1 is a major regulator of centrosome, we carried out immunofluorescence staining of γ-tubulin, a specific marker for centrosomes. While morphology of centrosomes showed normal patterns in BaF3 cells, in cells treated with BI-2536, replicated centrosomes failed to part from each other, surrounded by M phase chromosomes. Conclusions By integrating results from prior phosphoproteomics analysis, kinase inhibitor screening, ex vivo therapy in mouse transplantation experiments and in vitro viability assay using human xenografts, Plk1 inhibitor BI-2536 was shown to have potent activity against CRLF2-rearranged B-ALL cells. Our result suggests future therapy for this subset of B-ALL could be directed against Plk1 and its associated signaling pathways. Disclosures: Tyner: Incyte Corporation: Research Funding.

scholarly journals Targeting USP47 overcomes tyrosine kinase inhibitor resistance and eradicates leukemia stem/progenitor cells in chronic myelogenous leukemia

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Hu Lei ◽  
Han-Zhang Xu ◽  
Hui-Zhuang Shan ◽  
Meng Liu ◽  
Ying Lu ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Progenitor Cells ◽  
Chronic Myelogenous Leukemia ◽  
Drug Targets ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Myelogenous Leukemia ◽  
Tki Resistance

AbstractIdentifying novel drug targets to overcome resistance to tyrosine kinase inhibitors (TKIs) and eradicating leukemia stem/progenitor cells are required for the treatment of chronic myelogenous leukemia (CML). Here, we show that ubiquitin-specific peptidase 47 (USP47) is a potential target to overcome TKI resistance. Functional analysis shows that USP47 knockdown represses proliferation of CML cells sensitive or resistant to imatinib in vitro and in vivo. The knockout of Usp47 significantly inhibits BCR-ABL and BCR-ABLT315I-induced CML in mice with the reduction of Lin−Sca1+c-Kit+ CML stem/progenitor cells. Mechanistic studies show that stabilizing Y-box binding protein 1 contributes to USP47-mediated DNA damage repair in CML cells. Inhibiting USP47 by P22077 exerts cytotoxicity to CML cells with or without TKI resistance in vitro and in vivo. Moreover, P22077 eliminates leukemia stem/progenitor cells in CML mice. Together, targeting USP47 is a promising strategy to overcome TKI resistance and eradicate leukemia stem/progenitor cells in CML.

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